Shiro Akiyama, Neil J. Gemmell, Jennifer A. Marshall Graves and Neil D. Murray
School of Genetics and Human Variation, La Trobe University

The platypus is distributed in eastern Australia, ranging from subtropical to alpine areas, but is believed to be a single species despite some morphological variation. Its status is officially lower risk (locally threatened), but in some agricultural and urban landscapes, the situation is more critical. Therefore, suitable conservation strategies, derived from genetic data on the mating system, population structure and geographical variation, needed to be developed. Samples from localities ranging from south Queensland to Tasmania, and including inland rivers and King Island, were used to assess the geographic biodiversity. We also sampled the Shoalhaven River population, where long-term ecological studies have been carried out, and Warrawong Wildlife Sanctuary to assess the mating system and population structure. Microsatellite analysis revealed that sneaking non-territorial males contribute to breeding and sub-population structuring within a stream, and also to gene flow between streams. There were small genetic distances between mainland populations, but Tasmania was genetically distinct. Surprisingly, King Is. appears to be a monomorphic mainland population. All these results are discussed in terms of conservation strategies of the platypus.

 
2. Circadian expression of the period gene in two fruit fly species with different mating time

Xin An, Keryn Wilkes, Yemima Bastian and Marianne Frommer
Fruit Fly Research Centre, School of Biological Sciences A12, University of Sydney, NSW 2006

The period (per) gene is important for the generation and maintenance of biological rhythms. It was therefore investigated as a candidate gene which might be involved in the mating time isolation between two very closely-related sibling species, Bactrocera tryoni and Bactrocera neohumeralis, true fruit flies of the family Tephritidae. Initially, the threonine-glycine repeat region, which shows extensive length polymorphisms between closely related Drosophila species, was investigated. In B. tryoni and B. neohumeralis, the Thr-Gly tandem array is reduced to just a pair of repeats. No length polymorphisms were found over this region across several individual flies of each species from various latitudinal locations. Any sequence differences proved to be in the form of single base substitutions only and segregated independently of both species and latitude. Subsequently, a cDNA sequence from the 3' end of the B. tryoni per transcript was cloned and was used to design primers which amplified per-specific fragments from both genomic DNA and cDNA of the two species. RT-PCR amplification of this region revealed circadian oscillation of per expression in heads and abdomens of both species, with no differences in the timing of maxima and minima. Sequence comparison of the per transcripts is proceeding, but it appears most likely that differences in mating time are maintained by a gene or genes acting downstream of the period gene and that the primary circadian oscillators have not played a role in the speciation of B. tryoni and B. neohumeralis.

3. A QTL for litter size in mice ? preliminary results

W. Arsenault, N.J. Maqbool, I.C.A. Martin, C. Moran and F.W. Nicholas
Department of Animal Science, University of Sydney NSW 2006

In the latest of several crosses between the standard inbred strain C57 and the fecund inbred strain IQ5, 630 F2 females have been recorded for reproductive performance during their first three parities. This paper reports the results of a preliminary study on the first 252 of these females. Average litter size among these females ranged from 4 to 18. The top and bottom 25 females were genotyped at five microsatellite markers for which previous results (from other populations) had suggested linkage to QTL for litter size. For each marker, the existence of a QTL for litter size was assessed via a chi-squared test for difference in marker allele frequency between the top and bottom groups. Four of the markers produced non-significant differences, but a marker very near the centromere on chromosome 10 (D10MIT188) showed a highly significant difference (P = 0.0088). In a subsequent study in yet another population, this same marker has shown a significant effect on litter size (Maqbool et al., 1998). Interestingly, this marker is closely linked to the gene for the oestrogen receptor, which has been used commercially as a marker for fecundity in pigs.

Maqbool, N.J., Moran, C., Nicholas, F.W., Silva, L.P. and Martin, I.C.A. (1998). Searching for growth and fertility QTL in mice. Proceedings of the 6th World Congress on Genetics Applied to Livestock Production 26: 461-464.

4. Further testing for a body-weight QTL on mouse chromosome 3

N.J. Maqbool, C. Moran and F.W. Nicholas
Department of Animal Science, The University of Sydney, NSW 2006

Imprinting regions have been reported on 10 out of 20 mouse chromosomes. Silva (1994) found evidence for a QTL for post parturient growth on chromosome 3 near D3MIT24 which seemed to behave as if imprinted. In order to follow up this preliminary evidence of imprinting, a total of 371 backcross mice were produced from the same two genetically divergent strains used by Silva, namely the small C57/BL6J and the large, rapidly growing Inbred Quackenbush Swiss line 5 (IQ5). These progeny included 280 double reciprocal backcross females and 91 single-reciprocal-backcross males. All animals were recorded for body weight starting from three weeks of age until they were 16 weeks old. The female backcross progeny were not mated. All 371 mice were genotyped for D3MIT24 and the data analysed using a simple GLM analysis. The results did not confirm the presence of any QTL for body weight and so precluded an investigation of imprinting. However it may be that the effect previously observed is restricted to post parturient growth and is not found for early stage growth in males or females. IQ5 females have an enormous growth spurt associated with pregnancy and the animals measured in this study were not given the opportunity to express this growth, having mature body weights much lower (36.5g) than in the previous study (49.5g), where each female had produced four litters.

Silva, L.P. (1994). PhD Thesis, The University of Sydney.

5. Molecular evolutionary genetics of the Caenogastropoda

Emma Beacham, Winston Ponder and Don Colgan
The Australian Museum, 6 College St., Sydney

Caenogastropods are the largest and most conspicuous of the four main extant gastropod clades and, having undergone a series of major adaptive radiations during the early and late Mesozoic and the early Tertiary, exhibit great diversity in morphology, size, diet, reproduction and habitat. This variation, together with their good fossil record, makes them good candidates for detailed investigation of the factors triggering explosive diversification. Such investigations require a solid phylogenetic framework. Unfortunately, to date, no robust phylogenetic hypotheses based on either molecular or morphological characters are available for the Caenogastropods as a whole.

Previous molecular studies at the Australian Museum were unsuccessful in providing such a phylogeny owing to extremely low rates of evolution in the two studied regions of the 28S rDNA and in non-synonymous changes in histone H3.

In this project, we are studying more rapidly evolving sequences to test the monophyly and relationships of the principal Caenogastropod lineages, with a view to understanding their molecular evolution. Presently, an average of 600 bases of the cytochrome oxidase II gene from 20 Caenogastropods and 8 outgroups has been sequenced. An average of 415 bases from domain III of the 12S rDNA has been sequenced for 18 Caenogastropods and 7 outgroups. The cytochrome oxidase data have an approximately clock-like behaviour, with a low index of dispersion, but the 12S rDNA data imply markedly non-uniform rates, even with the exclusion of two aberrant sequences. Kimura 2 parameter distances within the Caenogastropods range up to 0.45 for the 12S rDNA sequences and up to 0.30 for cytochrome oxidase, suggesting that the data will be suitable for analysis of the group's phylogeny.

6. Molecular phylogenetics of the Polychaeta

Sarah Brown1, Greg Rouse2, Pat Hutchings1 and Don Colgan1
1The Australian Museum, 6 College Street, Sydney 2000
2School of Biological Sciences, The University of Sydney, NSW 2006

Until recently, the Polychaetes have been, phylogenetically, the least understood of the major invertebrate clades. The group lacked a higher level classification. Moreover, its boundaries and evolutionary relationships were unknown. Its position with respect to the Oligocheata and the doubtful phyla "Vestimentifera" and "Echiura" being particularly uncertain. In 1997, Rouse and Fauchald (Zool. Scipta 26: 139-204) published the first cladistically-based classification of the polychaetes based on a morphological character set. Some of the main clades within the group were well-supported but the majority of basal nodes had very few synapomorphies. Further data are required for the development of a robust phylogeny of the group. DNA sequences are an obvious source of such data. To date, however, there is little sequence information other than preliminary studies of the Polychaeta's relationships with the Echiura and/or the Vestimentifera which include a few ingroup taxa!

In this project, we have surveyed histone H3 and U2 snRNA sequences from 32 polychaetes and 10 other taxa, including an oligochaete, an echiuran and a vestimentiferan. This represents the first molecular study covering the (morphologically-estimated) phylogenetic range of the polychaetes. The data are not sufficient to suggest a new overall hypothesis of the group's phylogeny, but are suitable for statistically-testing the Rouse and Fauchald hypothesis. Results of winning-sites tests suggest several modifications to the hypothesis, including a re-alignment in the sister-pairing of the three main clades. The results also concur with previous molecular studies in placing both the Vestimentifera and the Echiura within the polychaetes.

7. Genetic criteria for reserve selection

Denis O'Meally and Don Colgan
Evolutionary Biology Unit, The Australian Museum, 6 College Street, Sydney 2000

The importance of conserving the genetic component of biodiversity for the long-term evolutionary potential of a species is widely recognised by both scientists and resource managers. Yet guidelines are lacking for ranking the genetic priority of different geographic areas for incorporation in reserves according to the comprehensivess, adequacy and representativeness criteria of the state and national biodiversity strategies. The aim of this project is to formulate such guidelines by testing the applicability of alternative ranking metrics to genetic data from geographically isolated populations of reptiles in the north-western slopes of the Great Dividing Range in New South Wales.

Metrics to be tested include phylogenetic diversity, character-change probability, taxonomic distinctiveness and synthetic descriptors of distances and other population genetic parameters.

The project will comprise both protein electrophoretic and molecular componenets. To date, electrophoretic surveys have been performed for eleven nominal species, collected in two large-scale field trips to the area. Genetic variation was substantially more than expected with at least three new species-level taxa being discriminated - in Amphibolurus nobbi, Eulamprus quoyi and Egernia striolata and genetic isolates being identified in several others. Two areas, one around Bolivia Hill and the second in the Mount Kaputar National Park, have been identified as being significantly genetically distinct.

8. Molecular systematics of Sminthopsini - a multigene approach

Mark Blacket1, Carey Krajewski2, Agatha Labrinidis3,4, Brian Cambron2, Steve Cooper4 and Michael Westerman1
1School of Genetics and Human Variation, La Trobe University, Bundoora, Vic 3083
2Department of Zoology, Southern Illinois University, Carbondale, Illinois 62901-6501, USA
3Department of Genetics, University of Adelaide, Adelaide, SA 5000
4Evolutionary Biology Unit, South Australian Museum, Adelaide, SA 5000

The marsupial family Dasyuridae comprises three subfamilies - Sminthopsinae, Dasyurinae and Phascogalinae. Sminthopsinae has two constituent tribes - Sminthopsini and Planigalini. On the basis of morphological characters a number of distinct clades have previously been identified within the tribe Sminthopsini. We have obtained mitochondrial and nuclear gene sequences from all genera and all but one species of this tribe in order to investigate their evolutionary relationships. A number of analytical approaches consistently identified particular clades - Antechinomys laniger, ningauis and four groups of species of Sminthopsis. The composition of these groups, their inter-relationships and congruency with clades previously proposed on the basis of morphological data will be discussed.

9. Molecular ecology of the broad-headed snake (Hoplocephalus bungaroides)

Emma Burns
School of Biological Science, UNSW 2052

The broad-headed snake(Hoplocephalus bungaroides) is considered potentially vulnerable to extinction. To aid the formulation of an effective species recovery plan, a study of the molecular ecology of the broad-headed snake was undertaken in this thesis. The utility of five microsatellite markers to measure the level of genetic variation in broad-headed snakes was investigated. Five polymorphic microsatellite markers with PIC values ranging from 0.103 to 0.737 were generated. These markers were used to analyse levels of genetic variation in twenty-four individuals from three populations of broad-headed snake. Allelic diversity for each population ranged from 2.6 in the North population to 3.2 in the Northwest population. Levels of heterozygosity detected within the three populations ranged from 0.307 in the South population to 0.560 in the North population. All three populations were found to conform to Hardy-Weinberg expectations and there was no evidence to suggest the occurrence of inbreeding within populations at this point. Although this study was limited by small sample sizes, the results imply that translocation between populations should not be necessary at this time. Contingency analysis detected no significant differences in allele frequencies between the North and Northwest populations (P 0.025) and significant differences in allele frequencies between the Northwest and South populations (P < 0.025). F-statistics indicated genetic differentiation between the Northern (North and Northwest populations combined) and Southern populations (FST = 0.2035; RST = 0.0831, P = 0.041). These results suggest that the Northern and Southern populations of the broad-headed snake should be recognised and managed as distinct, demographically independent units (ie separate Management Units). However, I recommend that the preliminary findings of this study be further investigated using larger sample sizes, additional microsatellite markers and mtDNA analysis.

 
10. Genetic characteristics of remnant populations of Swainsona recta (Fabaceae)

Lejla Buza1 and Andrew Young2
1Botany and Zoology, Australian National University, Canberra, ACT 0200
2CSIRO Plant Industry, GPO BOX 1600, Canberra, ACT 2601

Swainsona recta occurs in native grasslands and open woodlands in SE Australia. Historically, S. recta ranged from the north western slopes of NSW south to north-east Victoria. Due to habitat loss, it is now restricted to 17 populations located near Wellington, Mudgee and in the Canberra region. Population sizes vary between 10 and ~430 individual plants. To investigate the effects of population size on genetic diversity and levels of inbreeding in S. recta, allozyme electrophoresis was conducted on all remnant populations from seed collected in 1994 and 1997. Furthermore, to assess the effects of inbreeding on fitness, progeny representing three size classes (small, medium and large) were grown in a common environment and several fitness characteristics measured. Results show that smaller populations have less genetic variation than larger ones, are more inbred, and have reduced levels of fitness. This suggests that small populations are of limited value for in situ conservation of this endangered native pea.

 
11. Molecular and genetic options for resistance to organophosphorus insecticides

Zhenzhong Chen1, Emma Forbes1, Richard Newcomb2,3, John A. McKenzie1 and Philip Batterham1
1Genetics Department, University of Melbourne, Parkville 3052
2CSIRO Division of Entomology, GPO Box 1700, Canberra 2601
3Current Address: HortResearch, Private Bag 92-169, Auckland, New Zealand

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorus (OP) insecticides. OP resistance has developed via a range of mechanisms in insect species. Ace mutations have been identified in OP resistant strains of Drosophila melanogaster and Musca domestica. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field strains and laboratory generated mutants is determined by point mutations in the Rop-1 gene, which encodes an OP hyrolase, E3.

To investigate the bias for the Rop-1 /E3 mechanism in the evolution of OP resistance in L. cuprina, we have sequenced the Ace gene from this species. Five point mutations associated with reduced sensitivity to OP insecticides were identified in the Ace alleles from resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions which confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is due to factors other than codon bias. We expressed L. cuprina AChE in a baculovirus system to characterise its kinetic properties and its interaction with OPs. The most significant finding from these experiments is that wild type AChE is more than 30 fold less sensitive to OP inhibition than E3. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L.cuprina. As the primary target for OPs in this species, E3 serves to protect AChE. This capacity is enhanced by the Rop-1 mutation which increases the efficacy of E3 through enhanced OP hydrolase activity.

 
12. Syntenic linkage group shuffling: evolution of chromosome 3 in Lucilia cuprina

Jason Fair and Philip Batterham
Department of Genetics, The University of Melbourne, Parkville, Vic 3052

A combined molecular and genetic approach has been utilised to investigate the degree of evolutionary conservation within syntenic linkage groups of two higher Dipteran species, Drosophila melanogaster and the Australian sheep blowfly, Lucilia cuprina. While linkage groups appear to be conserved between the two species, this conclusion is based on a limited number of common markers and the degree of chromosomal rearrangement within syntenic units is yet to be determined. We have used the detailed molecular map of D. melanogaster to isolate and align several genes in L. cuprina that are homologous to X-linked genes of D. melanogaster. Following PCR, RFLPs have been detected between two isogenic strains in the L. cuprina orthologues of the D. melanogaster period (per), stress-sensitive B (sesB) and polehole (phl) genes. Initial genetic analysis with 5 phenotypic markers, indicate that all three molecular markers are associated with chromosome III of L. cuprina. However, intrachromasomal rearrangement during evolution has resulted in the shuffling of these genes relative to their locations on the X chromosome of D. melanogaster.

13. Coadaptation of the diazinon resistance system in Lucilia cuprina

Kris Freebairn, John A.McKenzie and Philip Batterham
Department of Genetics, The University of Melbourne, Parkville, Vic 3052

In the Australian sheep blowfly, Lucilia cuprina, resistance to organophosphorus insecticides (OPs) such as diazinon is due to a single amino acid replacement in the Rop-1 (R) gene product, resulting in a change of substrate specificity from esterase to OP hydrolase. Beyond resistance, flies carrying an R allele exhibit bristle asymmetry and decreased fitness in the absence of OPs. Continued use of diazinon after the evolution of resistance, selected for an unlinked fitness/asymmetry modifier gene/gene complex named Modifier (M).

Two possible molecular mechanisms have been proposed for the M. Firstly, the M gene may have a related function to R or, secondly, M may be involved in bristle formation. A specific form of the second hypothesis proposes M is an allele of Scalloped wings (Scl), the homologue of Notch in Drosophila melanogaster. Available genetic data are consistent with this. This hypothesis was further tested using a molecular approach based on the assumptions that the M allele of Scl arose once, recently. If these assumptions are correct, then a limited number of haplotypes would be expected for M alleles while a range of haplotypes may be expected for non-M alleles. Due to the size of the Scl gene, regions spanning the gene were examined using a combination of PCR size polymorphisms and DNA sequencing. Multiple haplotypes were found for M alleles. Therefore, either M is not an allele of Scl, or one of the assumptions is incorrect. Further mapping of M was done to further test this using a combination of RFLP and morphological mapping. It was found that M is not an allele of Scl.

 
14. Cytochrome P450s and insecticide resistance in the Australian sheep blowfly

Sarah Humphrey, John A. McKenzie and Phil Batterham
Department of Genetics, The University of Melbourne, Parkville, Vic 3052

Cytochrome P450s (CYPs) are a superfamily of monooxygenases capable of producing resistance to a wide range of insecticides through detoxification. CYP mediated resistance has been found in other diptera, including Drosophila melanogaster(Cyp6A2) and Musca domestica(Cyp6A1 and 6D1). In the Australian sheep blowfly, Lucilia cuprina , a rare resistant variant, Rop-2, was isolated in the 1970s. Preliminary work indicated CYP mediated resistance, but the strain was lost before this could be confirmed. Rop-2 mapped to chromosome VI which is syntenic to chromosomes 2R in D. melanogaster and 5 in M. domestica, the same chromosomes to which Cyp6A2 and Cyp6A1 localise.

This study aims to determine if CYPs are capable of producing insecticide resistance in L. cuprina. Genomic DNA fragments were generated following PCR with Cyp primers establishing that L. cuprina contains a complement of Cyp genes comparable to other insect species. The products of these clones show 30-60% amino acid identity with CYPs from other insect species. One clone appears to encode an orthologue to the Cyp6A1/A2 gene.

Elevated levels of CYP activity has been correlated with field resistance to the insecticide diflubenzuron in L. cuprina [1]. A field strain was selected for 4 years with diflubenzuron to generate a highly resistant strain [2] which exhibits low level cross resistance to other insecticides and increased CYP activity. The strain was obtained from Gary Levot for further study. Diflubenzuron resistance maps to a single locus on chromosome IV between the markers radial wing vein (ra) and tangerine eyes (tg). A cyromazine resistance gene also maps to this region [3] and could be allelic.

[1] Kotze, A.C. et al. (1997). Journal Economical Entomology 90:15-20.
[2] Kotze, A.C. and Sales, N. (1994). Journal Economical Entomology 84:355-360.
[3] Yen, J. et al. (1996). Australian Journal of Experimental Agriculture 36:413-420.

 
15. Genotypically dependent effects of cyromazine on reproduction and offspring development in the Australian sheep blowfly

Janet L. Yen, Philip Batterham and John A. McKenzie
Department of Genetics, The University of Melbourne, Parkville 3052

The insect growth regulator cyromazine is currently being used to control the Australian sheep blowfly, Lucilia cuprina (Wiedemann). While cyromazine resistance has not been observed in natural populations of L. curprina, resistant mutants have been isolated following EMS mutagenesis and selection in the laboratory. In this study the effects of cyromazine on egg production and subsequent egg-to-adult survival were examined on susceptible, heterozygous, and homozygous cyromazine-resistant genotypes ofL. cuprina, by administering 10 ppm of cyromazine to adults in drinking water. Cyromazine reduced egg production, hatch and subsequent larval survival in susceptible genotypes by acting at the embryonic stage of development. Resistance negated these effects dominantly for egg production and egg hatch and in a partially dominant manner for egg-to-adult survivorship.

 
16. Historical demography confounds inferences of contemporary population structure in the Indo-Pacific threadfin salmon, Polynemus sheridani

Steve Chenoweth and Jane Hughes
Griffith University, Brisbane, Queensland

An investigation of mtDNA control region variation in the estuarine threadfin salmon, Polynemus sheridani, throughout tropical northern Australia revealed significant population subdivision. Interestingly, pairwise comparisons involving the most distant estuaries were genetically homogenous despite significant structure at smaller spatial scales. This result implies either i) higher gene flow among distant locales than proximal ones or ii) a violation of the assumption of gene flow-drift equilibrium at large spatial scales. An investigation of these two competing hypotheses was conducted by reconstructing population histories for each oceanographic region sampled. Strong evidence was found for recent population expansions in the Gulf of Carpentaria and the Coral Sea. This was linked to sea level changes which altered the shape of estuarine habitat late in the Pleistocene. We propose that a large recolonising wave moved eastwards from the Timor Sea becoming established in the Gulf of Carpentaria once fully marine conditions were restored approximately 11000 ybp and subsequently the Coral Sea when the Torres Strait was last reopened by rising sea levels 7000 ybp. Thus sufficient time has not elapsed for the true demographic relationships among P. sheridani populations to register genetically at large spatial scales. Analyses which account for this historical confounding would be a benefit to researchers wishing to estimate demography from genetics in otherwise difficult to study marine populations.

 
17. Extreme length polymorphism of 18s rDNA within the scale insects

Lyn Cook
Division of Botany and Zoology, Australian National University, Canberra

 The small-subunit (18s) rRNA gene of Sternorrhyncha (white flies, psyllids, aphids and scale insects) is amongst the longest known for eukaryotes. A 5' region of the gene has been sequenced for the Australian gall-inducing scale insect Apiomorpha (Coccoidea: Eriococcidae). The fragment is about twice as long as the homologous region of other coccoid sequences. The increase in length is primarily the result of large insertions (up to 295bp), containing numerous dinucleotide and trinucleotide repeats, in three regions which are interspersed among highly conserved regions. The length of the 18s rDNA fragment, which is about 600bp long in other scale insects, varies from 760bp to 1169bp within Apiomorpha. A phylogeny derived from independent sequence data (COII) suggests that there is a trend towards increased length in 18s rDNA in more derived species of Apiomorpha.

 
18. Molecular phylogenetic analysis of the taxonomy of tephritid fruit flies (subfamily Dacine)

Sasha N. Curthoys and Marianne Frommer
School of Biological Sciences, Macleay Bldg A12, University of Sydney, NSW 2006

The family Tephritidae (true fruit flies) in the Order Diptera contains some serious horticultural pests. It has a morphologically based taxonomy which is unclear, especially for the subfamily Dacinae. Sequence data was analysed from 423bp of the nuclear period gene (exon 5); 500-1300bp of the nuclear scarlet gene (exon4-exon6, including introns); and 310bp of the mitochondrial cytochrome b gene. This data was acquired from both directions for three individuals each of 16 Dacine species, with a species from a different sub-family as the outgroup. DNA sequences were amplified using the polymerase chain reation and sequenced using cycle sequencing and dideoxy chain-termintaion. Phylogenetic relationships among the included taxa were inferred using both distance and maximum parsimony methods. These relationships appear largely consistent with the morphological classification. From this study it appears the period gene is inappropriate for phylogenetic studies, cytochrome b in the Dacinae is useful for a taxonomic level lower than genus, and the scarlet gene exon 5 is very useful at a genus and subgenus group level.

 
19. The evolution of parasitic strategies among the microgastroid Hymenoptera: evidence from multiple genes and morphology

Mark Dowton1,2 and Andrew D. Austin2
1Dept Biology, Wollongong University, Wollongong, 2522
2Dept Crop Protection, Waite Campus, Adelaide University, Glen Osmond, 5064

The microgastroids are a monophyletic complex of hymenopteran subfamilies that exhibit a diversity of parasitic lifestyles, although the biology of each subfamily is relatively uniform. This makes them an ideal model system for studying evolutionary transitions between variants of the parasitic lifestyle. The microgastroid subfamilies are all endoparasitoids of lepidopteran hosts, but can be solitary (lay a single egg per host) or gregarious (lay multiple eggs per host), oviposit into either the egg or larval stage of the host, and may attack caterpillars that live in concealed or exposed habitats. We investigated relationships among these wasps in order to trace evolutionary transitions between these various lifestyles. Analysis of 16S mitochondrial and 28S (D2 expansion region) nuclear rDNA genes, combined with morphological data, recovered a relatively robust phylogeny with all higher nodes of the tree supported by bootstrap proportions in excess of 70%. Mapping lifestyle traits onto this phylogeny revealed various evolutionary transitions. All basal lineages were solitary endoparasitoids, indicating that the ability to lay multiple eggs into a host is a recently acquired trait. The habit of ovipositing into the host egg is observed generally only in the Adeliinae and Cheloninae. These two subfamilies were recovered as a monophyletic assemblage, indicating a single, probably derived origin for this biology. Further, the basal lineages generally attack concealed hosts, suggesting that attack of more exposed hosts evolved more recently. Further, our phylogeny implies that certain of these lifestyle transitions are associated with rapid expansion of (extant) species numbers.

 
20. The ESU Status of Dasyurus maculatus, an endangered carnivorous marsupial

Karen B. Firestone
School of Biological Science, University of New South Wales, Sydney, NSW 2052
Conservation Research Centre, Taronga Zoo, P.O. Box 20, Mosman, NSW 2088

Tiger quolls, Dasyurus maculatus, are the largest carnivorous marsupials still present on the mainland of Australia, and occupy an important ecological niche. Populations of this species on the mainland and in Tasmania are fragmented. In addition, two recognized subspecies are geographically isolated, D. m. gracilis in north Queensland, and D. m. maculatus in the southeast of the mainland and Tasmania. Under the 1996 Action Plan for Monotremes and Marsupials D. m. gracilis is considered endangered while D. m. maculatus is considered vulnerable. The Evolutionarily Significant Unit (ESU) status of tiger quolls has not been examined previously, although they are actively managed. Ten populations of tiger quolls were sampled from throughout their range, including Tasmania and Queensland. Six nuclear microsatellite loci and the mitochondrial DNA (mtDNA) control region (507 bp) were used to examine the ESU status of this species. Results show that Tasmanian tiger quolls were reciprocally monophyletic to those from the mainland using mtDNA, but D. m. gracilis was not monophyletic to D. m. maculatus. Analysis of microsatellite loci also revealed significant differences between the Tasmanian and mainland tiger quolls, but not between D. m. gracilis and D. m. maculatus. These results indicate that Tasmanian and mainland tiger quolls form two ESUs but that D. m. gracilis and D. m. maculatus are part of the same management unit. These results indicate a need for a revised classification and management plan.

 
21. Presence of IAP determines the active state of the Axin (Fused) gene in mice

W.D. Flood1, T. Zhang2, F.D. Costantini2 and A. Ruvinsky1
1Department of Animal Science, University of New England, Armidale, NSW 2351
2Department of Genetics and Development, Columbia University, New York, NY 10032, USA

The Axin gene (formerly Fused) on Chromosome 17 causes vertebrae abnormalities and demonstrates inherited inactivation - reactivation in mice. The dominant effect of the mutant allele results from insertion of an IAP (Intracisternal A-Particle) element within intron 6, introducing a cryptic splice site. We investigated possible links between the presence of IAP and inherited inactivation?reactivation of Axin. The mutation tufted was used as a reliable genetic marker. In addition, two closely linked flanking microsatellite markers were used to trace possible recombination events. In observed inactive lines all animals had normal phenotype, IAP was absent and sequence of intron 6 did not differ from wild type. In two lines we found individuals with a reactivated allele, where IAP was present in the same position as the original mutation. Our observations indicate that inherited inactivation and reactivation of Axin correlates with presence or absence of the IAP element. On chromosomes bearing the inactive Axin allele, the proximal microsatellite marker D17Mit100 revealed two distinct variants which differ from both wild type chromosomes and chromosomes carrying the mutant Axin allele. Reactivation causes precise restoration of the microsatellite variant typical for chromosomes with the mutant allele. Modification of D17Mit100 due to inactivation?reactivation suggests that recombination is involved in movement of the IAP.

22. Genetic influence on caste in the ant Camponotus consobrinus

V.S. Fraser1, B. Kaufmann1, B.P. Oldroyd2, and R.H. Crozier1
1Department of Genetics and Human Variation, La Trobe University, Bundoora 3083
2School of Biological Sciences, University of Sydney, NSW 2006

The essence of advanced eusociality in certain Hymenoptera is the reproductive division of labor that exists between highly fecund queens and sterile or subfertile workers. A further division of labor (polyethism) can occur among workers such that individuals vary in their likelihood of performing different tasks. In some species of ants, polyethism is associated with morphological differences (i.e. workers and soldiers) (Oster and Wilson, 1978) and recently was shown by researchers to have a genetic basis in honeybees (eg. Robinson and Page, 1988) and (less clearly) ants (eg. Snyder, 1993).

The Australian sugar ant, Camponotus consobrinus, is an ideal organism for investigating the genetic basis to caste determination, because its workers show a triphasic allometry, differentiating into minors, medias and majors, each specialising at different tasks within the colony. Microsatellite analysis was carried out on workers from four polygynous C. consobrinus colonies to allow allocation of individuals to matrilines. Within colonies a relationship between size of an ant and her matriline was discovered, suggesting a genetic influence on caste determination.

Oster, G.F. and Wilson, E.O. (1978). Caste and ecology in the social insects. Princeton University.
Robinson, G.E. and Page, R.E. (1988). Genetic determination of guarding and undertaking in honeybee colonies. Nature 333: 356-358.
Snyder, L.E. (1993). Non-random behavioral interactions among genetic subgroups in a polygynous ant. Animal Behavior 46: 431-439.

1School of Science, University of Western Sydney, Nepean, PO Box 10, Kingswood, NSW 2747
2CRC for the Conservation and Management of Marsupials, Macquarie University, North Ryde, NSW 2109

Tumour necrosis factor (TNF) is a cytokine with a broad range of immunological actions. Two other cytokines, lymphotoxin alpha and beta (LT-a and LT-b), are structurally and functionally closely related to TNF. In eutherian mammals, the three genes for these proteins form a tightly linked gene cluster that resides in the major histocompatibility complex (MHC). In addition to the direct roles that TNF and LT play in immune and inflammatory responses, it has recently been shown in experiments with knockout mice, that they are also required for normal organogenesis and spatial organisation of lymphoid tissue. Marsupials split from the eutherian lineage approximately 130 million years ago, a sufficient time span for significant immunological divergence to have occurred. We are interested in defining key aspects of the immune system of a model marsupial, the tammar wallaby (Macropus eugenii) and have begun to clone a number of genes of immunological importance from this species. The present paper reports the cloning and characterisation of the TNF gene cluster in the tammar wallaby by RACE and genomic walking PCR.

 
24. The relative rate of DNA evolution in primates: new evidence from cats.

Genevieve Herbert1, Simon Easteal1, Stephen J. O'Brien2 and Leslie A. Lyons2
1The John Curtin School of Medical Research, The Australian National University, Canberra
2The National Cancer Institute, Frederick Cancer Research and Development Centre, Maryland, USA

Controversy remains over the issue of variation in the rate of nuclear genome sequence evolution among mammalian lineages. Data continue to be interpreted as indicating both uniformity and variation in rate. We have recently presented evidence of rate uniformity at 4-fold degenerate sites of 28 genes between rodents and primates [1], and at synonymous and nonsynonymous sites at 19 genes between apes (humans) and Old World Monkeys [2]. This appears to conflict with the findings of Ellsworth et al. [3]. To investigate this issue further within primates, we are comparing partial coding and noncoding sequences of 30 homologous genes in 15 primate taxa (three strepsirhines and twelve haplorines) using primers described by Lyons et al. [4]. We present preliminary results from these comparisons. This approach of using a large number of partial sequences from different genes avoids potential problems of bias due to effects of one or a small number of loci. In addition to the issue of relative evolutionary rates the data set provides a basis for addressing other issues including, phylogeny, divergence times and the relationship between variation in evolutionary rate in coding and noncoding regions among genes.

[1] Herbert and Easteal (1996). MBE 13:1054-1057.
[2] Easteal and Herbert (1997). JME 44 (Supp1):S121-S132.
[3] Ellsworth et al. (1993). Mol. Phylogenet. Evol. 2: 315-321.
[4] Lyons et al. (1997). Nature Genetics 15: 47-56.

25. Molecular evolution in Australian passerine birds

Margaret Heslewood* and Peter Baverstock
Centre for Animal Conservation Genetics, Southern Cross University, Lismore, NSW
* current address: Plant Molecular Systematics, School of Biological Science, University of New South Wales, Sydney, NSW

This poster will present an overview of the findings from an ongoing study looking at rates and patterns of molecular evolution in non-coding DNA. An analysis of DNA sequence data from 2 nuclear introns, myoglobin and aromatase, and the mitochondrial DNA control region will be presented for a range of passerines from the Parvorder Corvida including scrubwrens, thornbills, pardalotes and honeyeaters.

Despite an assumption of neutrality, differences are seen in the magnitude of sequence variation between taxa for the different loci, as well as in patterns of nucleotide variation and indel position within loci. These differences are possibly mediated by differences in mutation and/or selection although there are no known functions of these DNA regions which would place them obviously under selective constraints.

The potential of the different loci for population or phylogenetic applications will also be compared.

 
26. Evolutionary genetics of island populations of the Australian bush rat, Rattus fuscipes greyii, using microsatellite DNA analysis

Gavin Hinten1 and Peter Baverstock2
1Centre for Animal Conservation Genetics, Southern Cross Unversity, P.O. Box 157, Lismore, NSW 2480
2Graduate Research College, Southern Cross University, P.O. Box 157, Lismore, NSW 2480

The aim of this study is to use microsatellite DNA analysis to assess the extent and nature of genetic variation in island and mainland populations of the Australian bush rat, Rattus fuscipes greyii. Islands provide a natural laboratory for the study of microevolutionary processes. Studying isolated populations eliminates the potentially confounding effects of gene flow between populations. Moreover, in this study, the biogeographical history of the island populations is known, allowing calibration of molecular data with time. Mitochondrial DNA (Fiona Harriss, unpublished data) and MHC locus (Jenny Seddon, unpublished data) analyses have already been completed. Microsatellite DNA analysis would augment these previous studies, providing a comprehensive molecular analysis for these populations. Tissue samples have been collected from up to 30 individuals from each of 13 continental islands off the coast of South Australia. Twenty-two microsatellite primer pairs have been designed and initial primer screening has shown polymorphism. This study is still in progress, with results discussed during the presentation.

 
27. Polymorphism in Nramp gene and resistance to Johneís disease in ruminants

Ian Hughes1, Iain East2, and Daniel Sackey1
1School of Veterinary Science and Animal Production, University of Queensland
2CSIRO Tropical Agriculture, Long Pocket, Queensland

Johneís disease is chronic wasting disease affecting cattle, sheep, and goats. It is caused by infection with Mycobacterium paratuberculosis which colonizes and multiplies within the mucosal cells of the small intestine.

In mice, allelic variation of the Nramp (natural resistance associated macrophage protein) gene has been found to be associated with the ability to resist infection with a range of intracellular pathogens such as Mycobacterium spp., Salmonella typhimurium and Leishmania donovani. Nramp codes for an intracellular-membrane channel that is found primarily in macrophages. Nramp is an important factor in phagocytosis and is thought to pump essential-metal ions away from bacteria trapped in phagosomes.

Variation in resistance to Johneís disease exists within each of the ruminant species. We are currently looking for sequence variation in the cDNA of Nramp from cattle, sheep and goats that may correlate with this variation in resistance. A comparison of allele frequencies between infected and non-infected animals will then be undertaken. Infection in individuals from Johneís affected farms, rather than clinical signs, is used as the relevant phenotype as Nramp is only associated with the initial stages of resistance to infection with M. paratuberculosis.

In work already completed, a number of cDNA-sequence variations have been detected in the bovine Nramp gene. Three of these have been identified in more than one animal and confirmed by sequencing in both directions.

28. Hemeralopia (day blindness) in Alaskan Malamute dogs

Ian Hughes and Edith Hampson
School of Veterinary Science and Animal Production, University of Queensland

Hemeralopia or day blindness is a condition that particularly affects Alaskan Malamute dogs. Affected dogs behave normally in dim light but demonstrate severe loss of vision in bright daylight. The condition is caused by the specific degeneration of cone cells in the retina. In bright light normal rod cells cease functioning and vision occurs via the cone cells. Thus, bright light renders hemeralopic dogs blind.

Hemeralopia has been reported to be inherited as an autosomal recessive with affected dogs showing clinical signs between 8 weeks and 7 months. Definitive diagnosis involves recording the electrical response of cone cells to a light stimulus by electroretinography (ERG). Specifically, a response to a 30Hz flicker is specific for cone function with hemeralopic dogs showing a flat line.

We are investigating an affected Malamute pedigree. DNA samples from over 40 dogs have been obtained and 17 dogs have been ERGd. A detailed pedigree analysis and segregation analysis with putatively linked microsatellite markers are yet to be performed. However, some initial observations are worthy of comment. First, the age of onset appears to be later in these dogs, both in behavior, and in development of a flat-line ERG. Secondly, very few of the Malamutes have a perfect ERG (4 dogs of other breeds displayed a typically normal ERG). The ERGs of dogs from 8 months to 6 years of age showed considerable variability in severity. While we will present further analyses, these initial observations suggest that the inheritance of this disease may be more complex than has been generally recognized.

 
29. Genotype by environment interaction for milk yield in Australian Holstein dairy cattle

M. Montes-Castillo and I.P. Hughes
School of Veterinary Science and Animal Production, The University of Queensland, St Lucia QLD 4072

Genotype by Environment Interaction (GxE) can be recognized as heterogeneity of variances, change in genotype ranking from one environment to another, or both. It is possible that GxE with respect to geographical location is more important than currently appreciated in Australia, given the marked variation in the climate and physical environment between states. A study was therefore conducted to evaluate the significance of genotype by environment interaction in milk production for Australian Holstein-Friesian cattle in Queensland and Victoria.

First lactation records from 111,165 cows calving over an eight year period (1987-1994) were examined. 644 sires were considered as genotypes. Milk yield records were analyzed over the two states, considered as two different environments. An Estimated Breeding Value (EBV) for milk yield was determined for each sire in Queensland and Victoria based on their daughters performance in those states. An animal model of BLUP was used in these calculations. Spearman Rank Correlation between these EBVs for Queensland and Victoria was calculated to be 0.30, suggesting a significant GxE interaction.

It is concluded that the relative superiority of sires in temperate environments may not be maintained in the tropics and subtropics because of the GxE. Further analyses are being made and the nature of the suggested GxE investigated.

 
30. Living with a genetic disorder

Sarah Jordan and Lynette McLean
Division of Animal Science, University of New England, Armidale, NSW 2351

This is a preliminary investigation into some of the clinical and social effects of three genetic disorders in humans: Retinitis pigmentosa (RP), Cystic fibrosis (CF) and Darier's disease. A literature review followed by surveys and discussions with several affected individuals and people from state support groups is being undertaken this year. The aim of the investigation is to determine how suffering from a genetic disorder affects an individual's life and the impact on the family. Cystic fibrosis is an autosomal recessive complaint affecting 1 in 2,000 newborn in Australia. It primarily affects the exocrine system and current work on treatment includes the use of genetic therapy. Retinitis pigmentosa transmission is complicated by the fact that in different families it can be inherited as an autosomal dominant, autosomal recessive or an X-linked trait. This disorder affects the retina and leads to blindness. The frequency of sufferers in Australia is unknown. Darier's disease is caused by an autosomal dominant allele which shows variable expressivity and partial penetrance within a given family. This primarily affects the skin by inducing itchy, scaly, red-brown pigmented papules that can lead to extremely uncomfortable plaques. These often appear on the torso, face and extremities. The present support groups in Australia are quite strong for CF and RP, however no known group exists for those suffering from Darier's, nor any skin disorder in general. This study will provide information to support genetic counselors in offering holistic support for their clients.

 
31. A Poisson GLM application to QTL analysis of discrete backcross data using the E-M algorithm

S.A. Kayis1, P.C. Thomson1 and F.W. Nicholas2
1Biometry Unit, Dept. of Crop Sci., University of Sydney, NSW 2006
2Department of Animal Science, University of Sydney, NSW 2006

Many methods have been developed for detecting quantitative trait loci (QTL). However, these are almost always based on a normal distribution of the trait, and therefore are not suitable for non-normal data such as litter size, calving ease etc.

Kayis et al. (1998) presented a single-marker-single-QTL method, estimating recombination between the marker and QTL with associated QTL effects, for analysing QTL data for litter size in backcross mice - a discrete variable. In this study, a Poisson model for litter size is assumed within the framework of a generalised linear model. Parameters (bi, i = 1,...,4; gk, k = QQ, Qq, qq, qQ; b : parity effect; g : QTL effect) were estimated via the E-M algorithm for each fixed QTL position. When the putative QTL is located between two genetic markers, a flanking marker model is used. Likelihood-ratio-type confidence intervals are obtained for putative QTL location. Statistical aspects of this study have been submitted to the 14th Australian statistical conference.

The method has been applied to backcross mice data, kindly provided by Silva (1994). Results showed some evidence for QTL, affecting litter size, on different chromosomes.

Silva, L.P. (1994). "Genetic Analysis of Litter Size and Body Weight in Mice". PhD Thesis, University of Sydney.
Kayis, S.A., Thomson, P.C. and Nicholas, F.W. (1998). QTL Analysis of Discrete Backcross Data Using the E-M Algorithm. Proceedings of the 6th World Congress on Genetics Applied to Livestock Production.

W.Y.N. Man and F.W. Nicholas
Department of Animal Science, University of Sydney, NSW 2006

In recent years, there has been considerable interest in the effect on Australian thoroughbreds of the use of so-called "shuttle" stallions ? northern-hemisphere stallions that are flown to Australia each year, to be mated with local mares. In order to investigate the genetic effect of these stallions, work has commenced on a pedigree analysis of the almost 200,000 Australian-born thoroughbreds whose pedigrees have been recorded by the Australian Stud Book (ASB). A preliminary analysis by Vaez Torshizi et al. (1998) showed that the average annual rate of inbreeding from 1813 to 1996 has been approximately 0.00057, culminating in a current level of inbreeding of around 0.09. Like all traditional pedigree analyses, the above results are underestimated because of incomplete pedigree information. These limitations have been partly overcome by the development of parameters such as the effective number of founders, the effective number of ancestors, and the effective number of founder genomes. This paper presents estimates of these parameters for Australian-born thoroughbreds, together with an estimate of the similarity of sub-populations, which collectively provide greater insight into the genetic structure of this population, and the likely impact of shuttle stallions.

Vaez Torshizi, R., Nicholas, F.W. and Tier, B. (1998). Inbreeding in Australian Thoroughbred horses and the implications of "shuttle" stallions. Proceedings of the 6th World Congress on Genetics Applied to Livestock Production 24: 440-443.
 

33. Hereditary zinc deficiency in Angus and Holstein-Friesian cattle: a homozygosity mapping approach for the localisation of the causative gene

I. Tammen1, H.W. Raadsma1, R.W. Cook2 and F.W. Nicholas3
1University of Sydney, Department of Veterinary Clinical Science, Camden, NSW
2NSW Agriculture, Wollongbar, NSW
3University of Sydney, Department of Animal Science, Sydney, NSW

Hereditary zinc deficiency is a lethal genetic disorder with an autosomal recessive mode of inheritance. The condition was first observed in Holstein-Friesian cattle and described as lethal trait A46. More recently it was diagnosed in a herd of Angus cattle in Australia. The condition is considered to be homologous to an inherited human zinc deficiency syndrome, Acrodermatitis enteropathica.

Our aim is to obtain a chromosomal localisation of the recessive disease loci in both Angus and Holstein-Friesian cattle. To maximise use of limited pedigree material, and minimise the total genotyping effort, homozygosity mapping is being utilized in combination with DNA pooling. Markers of interest will be validated by traditional linkage analysis in the enlarged families. In addition, we exploit information of putative candidate genes from the human/mouse map [e.g. cysteine-rich intestinal protein (CRIP), metallothionein (MT), and zinc transporter 1-4 (ZnT1-4)] to increase marker density on homologous chromosomal regions of these genes on the bovine map. This allows us to increase the power of homozygosity mapping with marginal increase in genotyping effort.

34. Hsp68 variation in the genus Drosophila

Mark Kellett, Gawain McColl and Stephen W. McKechnie
Department of Biological Sciences, Monash University

It has been known for some time that pretreatment of an organism with an elevated, but non-stressful temperature can increase its resistance to subsequent heat stresses. This phenomenon is known as the heat hardening response. Recent studies in our laboratory involved selection of populations of D. melanogaster under differing regimes of thermal stress. Resultant changes in allele frequencies suggested a link between the heat shock protein 68 gene (hsp68) and the levels of hardened heat resistance. This result prompted the sequencing of the hsp68 gene.

In current experiments, we are attempting to determine if this association between hsp68 and the heat hardening response also exists at the inter-specific level. The D. melanogaster sequence was used to create a set of primers that would amplify the coding region of hsp68. These primers have been used to amplify PCR product from 3 species from the melanogaster sub-group (D. mauritiana, D. yakuba and D. erecta) and 3 members of the montium sub-group (D. auraria, D. serrata and D. vulcana). Sequencing of these PCR products has revealed that hsp68 is conserved between these species, though some variation is present in the 3' region which encodes the substrate binding domain. It is also apparent that hsp68 is more diverse among members of the montium sub-group than among members of the melanogaster sub-group. Future experiments will involve quantifying the heat hardening response of these species to look for associations with variation in both the expression pattern and the sequence of hsp68.

 
35. A novel Bovidae interspersed DNA repeat isolated from sheep

Helena Kojevnikoff, Jan Cook, Graham C. Webb and Cynthia D.K. Bottema
Department of Animal Science, Waite Campus, University of Adelaide, Glen Osmond, SA 5064

Mammals have several different types of repetitive DNA families within their genomes, including dispersed repeats such as long and short interspersed nuclear elements (LINES and SINES). In trying to understand the role of these repetitive DNA sequences in the function and evolution of Bovidae genomes, we have taken several approaches to clone the repeats from cattle and sheep. One such approach is to isolate specific fragments produced by digestion of genomic DNA with certain restriction enzymes.

Upon restricting sheep genomic DNA with Pst I, we observed a 2 kb band. The band was cloned and used as a probe for Southern analysis of sheep genomic DNA digested with other restriction enzymes. A smear was observed on the Southern blots for all the restriction enzymes, suggesting that the clone might represent a DNA repeat. Consequently, the sheep clone was also used as a probe in fluorescence in situ hybridisation (FISH) experiments against sheep and cattle metaphase chromosomes. Scattered grains were observed on all the sheep chromosomes, and thus, the repetitive nature of the clone was verified. There also must be an equivalent cattle repeat since the sheep clone cross-hybridised to bovine chromosomes. Because few grains were observed, the sequence is not as common as other identified Bovidae repeats such as BovA, BovB or BDF. The clone was sequenced, but no extensive homology was found with any other known repeats or genes. It can be concluded that a new Bovidae interspersed repetitive DNA family has been discovered.

 
36. Localisation of centromeric satellite sequences in cattle Robertsonian translocations

Jianze Zheng, Graham C. Webb, Helena Kojevnikoff and Cynthia D.K. Bottema
Department of Animal Science, Waite Campus, University of Adelaide, Glen Osmond, SA 5064

There are 4 major classes of cattle centromeric satellite DNA sequences. Satellites I and II hybridise to the procentric regions of all cattle autosomes, while Satellites III and IV hybridise to different subsets. We analysed the pattern of localisation of each of these satellites in relation to each other using two-colour fluorescence in situ hybridisation (FISH).

We observed that the amount and location of the satellites vary between the chromosomes. However, some generalisations can be drawn. For instance, our results suggest that Satellite III is on the long arm of autosomes, while Satellites I, II and IV are more on the short arm. Satellites I, II and IV frequently appear to overlap, although usually Satellite IV is more p-terminal than Satellite II and Satellite II is more p-terminal than Satellite I. The conclusion is that these satellites may be interspersed and not present as single isolated blocks.

We also analysed the distribution of these satellites in two cattle Robertsonian translocations, the monocentric t(1;29) and the dicentric t(14;20). Based on the localisation of the satellites on chromosomes 14 and 20, the t(14;20) translocation can be explained as a simple fusion of the two centromeres with little or no loss of satellite sequences. However, based on the localisation of the satellites on chromosomes 1 and 29, no simple model can explain the formation of the t(1;29) monocentric fusion. In fact, the t(1;29) appears to have evolved to its present state with an unexpected loss and rearrangement of satellite sequences.

 
37. Comparative porcine gene mapping in relation to human chromosomes 9, 10, 20 and 22

J.H. Lee1, W. Zhang1, Y. Chen1, L. A. Lyons2, A. Robic3 & C. Moran1
1Dept of Animal Science, University of Sydney, NSW 2006
2Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702-1201, USA
3INRA, Laboratoire de Génétique Cellulaire BP 27, 31326 Castanet-Tolosan Cedex, France

Comparative gene mapping is facilitated by consensus PCR primers which amplify homologous gene fragments in many species. We used 53 CATS (Comparative Anchor Tagged Sequence) consensus primer pairs for loci located on human chromosomes 9, 10, 20 and 22, for amplifying homologous loci in pigs. 48 primer pairs have yielded PCR products which have been sequenced. The identities of 23 CATS products have been confirmed by comparison with homologous sequences in GenBank. However only 2 of these have yielded polymorphisms permitting linkage mapping. A French somatic cell hybrid panel has been used to physically map 11 porcine CATS products distinguishable from rodent background product, namely ADRA1A, ADRA2A, AMBP, ARSA, EGR2, GNAS1, GRP78, IFNB1, SPTAN1, TOP1 and TXN. ADRA1A maps to SSC16, a position more consistent with its original localisation on HSA5q rather than its reassigned human position on HSA20, but the porcine CATS product also shows high similarity to human ADRA1B, located on HSA5q. Other loci map to positions consistent with known syntenic relationships between human and pig. Despite low levels of polymorphism, frequently indistinguishable rodent and porcine products in somatic hybrids and some confusion of identity of gene family members, the use of consensus primers has made a useful contribution to the porcine human comparative map.

 
38. QTL mapping on chromosome 2 in pigs using F2 families from Meishan, Piétrain and Wild Boar

S. Lee1, G. Moser1,2, Y. Chen1, E. Müller2, H. Geldermann2 & C. Moran1
1Dept of Animal Science, University of Sydney, NSW
2Dept of Animal Husbandry and Breeding, Division of Animal Breeding, University of Hohenheim, Stuttgart, Germany

The Meishan x Piétrain and Wild Boar x Piétrain F2 pedigrees developed at the University of Hohenheim have been or are being scanned for putative QTLs on chromosome 2 for 43 performance traits using S0141-Sw240-MYOD1-Sw395-S0010. These markers are from 10 to 20cM apart and cover ca. 50% of chromosome 2. QTLs were mapped using a multiple marker least squares regression method and empirical significance thresholds were obtained using permutation tests. The analysis of the Meishan x Piétrain pedigree detected QTLs affecting 11 traits at the 5% chromosomal significance level, each accounting for 3.3 to 7.5% of the variance in the F2 generation. QTLs for carcass traits, such as lean to fat ratio, back fat weight and weight of shoulder meat were mapped in the region 23cM-42cM. Meishan alleles were associated with increased fatness of the carcass and lower weights of lean cuts. A QTL for pH, 24h p.m. in M. long. dorsi, with a relatively large dominance effect and accounting for 3.9% of F2 variance, was mapped at 38cM. A large effect on number of teats, accounting for 6% of the F2 variation, mapped to 6cM. Three of the mapped QTLs (number of teats, lean cut percentage, bacon meat/half carcass weight) are also significant at the approximated 5% genome-wide significance thresholds, and it is reasonable to assume that the effects found are real. It will be interesting to see if any of these QTLs are found in the Wild Boar x Piétrain pedigree.

 
39. Analysis of regulatory sequences for multiple environmental signals using reporter gene fusions

Callum A. Mack and Margaret E. Katz
Division of Molecular and Cellular Biology, School of Biological Sciences, University of New England, Armidale, NSW 2351

The expression of Aspergillus extracellular proteases has been shown to be regulated by least five environmental signals (carbon, nitrogen and sulphur metabolite repression, pH control and induction by exogenous protein). Two extracellular protease genes have been isolated from Aspergillus nidulans. The prtA gene encodes an alkaline (serine) protease and the prtB gene encodes an acid (aspartic) protease. To localise specific regulatory sequences controlling the response to each environmental signal, sections of the 5' region of the prtA gene have been fused, in frame, to the E. coli lacZ reporter gene.

Gene fusions containing from between 85-535bp of sequences upstream from the translation start point have been constructed and introduced, via transformation, into an A. nidulans strain that produces wild type levels of extracellular protease. Each construct will also be tested in strains which carry mutations affecting extracellular protease production. Preliminary results suggest that gene fusions containing 350bp upstream from the prtA start codon produce high levels of b-galactosidase in response to carbon, sulphur and nitrogen limitation and, therefore, must contain the regulatory sequences for these three environmental signals.

 
40. Production of a preliminary genetic sexing strain in the Queensland fruit fly, Bactrocera tryoni

P. Maheswaran, J. A. Sved, M. Frommer and A. Meats
Fruit Fly Research Centre, School of Biological Sciences A12, University of Sydney, NSW 2006

This research was aimed at obtaining visible markers and translocation strains that will be useful in producing a male-only strain for sterile insect technique (SIT) in the Queensland fruit fly, Bactrocera tryoni. Visible mutations ? white-marks (wm), droopy-antennae (dr), bent-wings (bw), and three allelic eye colour mutations, orange-, lemon-, tomato-eyes (oe) ? have been isolated. The white-marks mutation may be useful as a self-marked release strain. Flies with the bent-wings mutation have a kink in their wings, which renders them unable to fly. Also, the bent-wings strain has been found to show a temperature-sensitive lethal phenotype. The eye-colour markers appear to be mutations of a gene homologous to the Drosophila melanogaster scarlet and Lucilia cuprina topaz genes. If so, this gene may be useful as a marker for gene transformation.

Four Y-autosome translocations involving the white-marks mutation on chromosome 2 have been produced and one remains extremely stable and robust. Recently, two translocation strains involving the bent-wings mutation (a deleterious marker) have been produced. Further studies are in progress to determine a suitable temperature that would eliminate the female flies in rearing for sterile release.

 
41. Genetic and physical chromosome maps of the Queensland fruit fly, Bactrocera tryoni

Jing-Ting Zhao, Marianne Frommer and John Sved
Fruit Fly Research Centre, School of Biological Sciences A12, University of Sydney, NSW 2006

Polytene chromosomes from salivary glands have been studied for the Queensland fruit fly, Bactrocera tryoni, leading to a physical chromosome map. Substantial chromosome synteny exists with the chromosome set of the Mediterranean fruit fly, Ceratitis capitata. The same five autosomal chromosomes have been identified, with evidence for both pericentric and paracentric inversion events in the divergence of the two species. Several cloned genes have been placed on the two chromosome sets by in situ hybridisation.

DNA microsatellites have enabled us to map visible mutations. Three-generation pedigrees involving particular visible mutations are typed for microsatellite variants by PCR analysis. Although it is not easy to construct strains with known microsatellite genotypes, we have found that the level of microsatellite heterozygosity is sufficiently high, so that a substantial proportion of such three generation families are informative in mapping of mutations and microsatellites. The single-copy DNA sequences flanking variable microsatellites have been located on polytene chromosomes using in situ hybridisation, thus providing a link between the genetic and physical maps. The current integrated genetic and physical map of B. tryoni is presented.

 
42. Genetic analysis of the LKB1 gene in a large Peutz-Jeghers Syndrome family

Mary McPhillips1, David Mowat2, Cliff Meldrum1, Allan D. Spigelman1 and Rodney J. Scott1
1John Hunter Hospital, New Lambton, NSW 2305
2Hunter Genetics, Waratah, NSW 2289

Peutz-Jeghers Syndrome (PJS) is an autosomal dominant disorder characterized by the development of haematomatous adenomas occurring at various sites throughout the gastrointestinal (GI) tract and pigmental abnormalities found especially in the buccal cavity. Persons afflicted with this disease have a very much greater risk of developing neoplasia especially cancers of the GI tract but they also have an increased risk of developing cancer at other sites, most notably pancreas, breast, ovary and gallbladder. The disease appears to be genetically heterogeneous as at least two sites have been identified which segregate with the disease in different families. Disease penetrance is estimated at being virtually 100%. We have identified a large Peutz-Jeghers Syndrome family where there are 9 affected individuals crossing two generations. Linkage analysis using the markers D19S565, D19S878 and D19S886 has revealed that the disease locus is on chromosome 19p13.3 and that there are several nonaffected persons in the second generation, suggesting that penetrance is not 100%. Sequence analysis of the LKB1 gene will be described that indicates more precisely the level of disease expression which casts doubt on the high degree of penetrance previously reported by others.
 

43. Collie Eye Anomaly, a dog congenital defect

Elisa Mokany and Alan Wilton
School of Biochemistry and Molecular Genetics, University of NSW, Sydney 2052

Inbreeding in dogs has lead to a large number of heritable diseases that are prominent in certain breeds. Eye defects are amongst the most common. Collie eye anomaly (CEA) is an autosomal recessive disease which occurs at a very high incidence in collie breeds. The disease has a variable expression with characteristics ranging from blindness, caused by detached retina, to mild choroidal hypoplasia which has no affect on sight.

We aim to find a linked marker to CEA so that a DNA based test can be developed for carriers of the defective gene. The breeders can then use this to eliminate the disease by selective breeding. This is also the first step in identification of the disease gene which will help elucidate the development process of the eye.

The dog genome screen was started by typing border collie pedigrees for 26 randomly chosen highly polymorphic microsatellites. LOD score analysis showed no significant evidence of linkage of the microsatellites to CEA and close linkage was excluded from 15 microsatellites. Suggestions of linkage were found but for significant results the power of analysis needs to be increased by including more individuals to the pedigree.

The linkage between microsatellites was checked against a recently published dog map.

44. Molecular comparison of Australian dingo and domestic dog

Alan Wilton, David Steward and Katina Zafiris
School of Biochemistry and Molecular Genetics, University of NSW, Sydney, NSW 2052

The dingo probably arrived in Australia only 5,000 years ago. It is closely related to domestic dogs and is thought by some to be just a wild domesticated dog from Asia. The dingo does have several morphological, behavioural and reproductive characteristics that differentiate it from domestic dogs, eg. annual breeding cycle. Although classified as a noxious pest by most government agricultural agencies, it is the source of a conservation campaign. The dingo as a pure breed is in danger of extinction because of hybridisation with wild domestic dogs. Skull morphometrics and other physical characters have been used to attempt to distinguish hybrids but it is not a reliable method.

We are looking for molecular markers that will allow differentiation between dingos and domestic dogs. We have examined 50 canine microsatellites and all amplify in the dingo. Only one produced a large number of null alleles. Most show overlapping allele frequency distributions. Some are sufficiently different to be used in an assessment of purity on probability estimates. One locus has been identified which is diagnostic for dog or dingo origin. An estimate of 10 diagnostic loci are needed to reliably ensure hybrid backcrosses are removed from breeding programs. Other markers such as sequence differences in introns such as those among Comparative Anchor Tag Sites and mitochondrial DNA markers are also being investigated. Screening of a larger set of genetic markers is likely to produce diagnostic markers that can be used in the preservation of the dingo.

 
45. Homologous structures, homologous genes: where's the constraint?1

Valerie B. Morris
School of Biological Sciences A12, University of Sydney, NSW 2006

In the time before Genetics, if ever there were one, homology was decided by visible likenesses. In the time after Genetics, which is now upon us, homology is decided by sequence likenesses. So who shall prevail? Or, will it be that another shall lead us into the light?

For it is true that in the beginning when genes created form, form and genes were equal, so from whence came forth the constraints that created the inequality that is around today?

Using examples, I consider some matters relevant to the meanings we presently attach to homology.

1Dickinson, W.J. (1995). Molecules and morphology: where's the homology? Trends in Genetics 11: 119-121.

 
46. The use of an isogenic seed line and differential display to search for the genes responsible for plant regeneration via somatic embryogenesis in the model legume, Medicago truncatula

Kim E. Nolan and Ray J. Rose
Department of Biological Sciences, The University of Newcastle, NSW 2308

Cultured plant cells have the capacity to express their totipotency by producing embryos from somatic cells. Somatic embryogenesis is used as a model system to study plant embryo development, but the genetic induction of embryo development remains elusive. Medicago truncatula is a model legume for the study of nitrogen fixation. One cultivar, Jemalong, can be regenerated via somatic embryogenesis but very few embryos form on the cultured tissue. Regenerated Jemalong plants have the capacity to produce greatly increased numbers of somatic embryos when tissue from these plants is put through another cycle of tissue culture. This increased embryogenic capacity is inherited through the seed. Selection of the plants producing the highest embryo numbers over several generations has enabled the development of a highly embryogenic seed line called "2HA". 2HA is an isogenic line of Jemalong, with the only known difference between them being 2HA's increased embryogenic capacity. This makes it an excellent candidate for the study of embryogenic genes. The two lines can be cultured under identical conditions, but 2HA will form numerous embryos whereas Jemalong forms very few. If differences between the messenger RNAs the two seed lines produce at the time of embryo induction can be found, it should be possible to identify embryo induction genes. We are currently using differential display to search for such differences in gene expression.

 
47. Optimal gene/sequence combinations for determining phylogeny from public sequence databases

Tania Oh, Jenny Saleeba, Ben Oldroyd and Tim Littlejohn
School of Biological Sciences, University of Sydney

Public databases of DNA sequences are used to deposit virtually all sequence information obtained in laboratories throughout the world. Sequence records are required to contain taxonomic information about the organism from which the sequence was obtained. Prima facae these records should represent an outstanding resource for phylogenetic studies. However, retrieving useful phylogenetic information from the databases is often difficult because global sequencing efforts are uncoordinated. This means that the same gene is unlikely to have been sequenced for all the species of interest. Computer tools are therefore required to determine what gene/species combination will yield the greatest phylogenetic information.

It is anticipated that this bioinformatics project will develop a computer program to improve methods for retrieving phylogenetic information. The program would identify DNA sequences, for a user-defined search space, with the highest amount of phylogenetic information for the group being investigated. For example, a query such as "what homologous sequences are available for fungi, that could be used to construct phylogenetic inference?" Valuable resources would be saved as the biologist will be able to determine which sequence yield the highest amount of phylogenetic information without having to generate these sequences in the laboratory.

The preliminary design of such a computer tool will be presented. The algorithm which rates the relative value of sequence groups for phylogenetic analysis will be discussed and the tables needed to provide all necessary information will be shown.

48. The HRAS1 minisatellite in an Aboriginal Australian population

June Roberts-Thomson1, Rodney Scott1 and Barry Boettcher2
1John Hunter Hospital, Newcastle, NSW
2Dept. Biological Sciences, Newcastle University, NSW

There is a high incidence of disease in Aboriginal Australians compared to Caucasians. Minisatellites have been demonstrated to be associated with a number of diseases, such as cancer, diabetes and cardiovascular disease. Minisatellites have high heterozygosities and population differences have been observed in both allele number and frequency distributions. Previous studies on Aboriginal populations have observed a low genetic diversity at protein loci and the alpha-globin gene complex. However the same low diversity is not consistently observed with minisatellites.

The HRAS1 minisatellite has been widely studied in Caucasians, where the heterozygosity is 65% and rare alleles have been shown to be associated with a number of different cancers. In an Aboriginal population from central Australia, the heterozygosity was 72%. Internal mapping techniques were used to look at the internal structure of the HRAS1 VNTR, by utilizing the presence of infrequent HaeIII restriction sites. Partial digest techniques applied to the 30-repeat common allele identified similar restriction patterns in the HRAS1 VNTR in the Aboriginal population, unlike the Caucasians studied. This suggests a single lineage for the allele in the Aboriginal population. By comparing the internal maps of different HRAS1 minisatellites, and from sequence data, possible relationships between different alleles will be discussed.

 
49. Low genetic variation at microsatellite loci in a critically endangered New Zealand parrot

Bruce C. Robertson, David M. Lambert and Ed O. Minot
Ecology, Institute of Natural Resources, Massey University, Private Bag 11-222, Palmerston North, New Zealand

The kakapo Strigops habroptilus is a large (c. 2 kg), flightless, herbivorous, nocturnal parrot that displays a lek mating system. Numbering only 57, kakapo are currently the focus of intensive conservation efforts. As part of these efforts, microsatellite DNA markers were required to obtain information on the relatedness and parentage of surviving and recently deceased birds. Potentially, these markers would allow us to identify individuals from small amounts of material such as faecal samples. We isolated kakapo (CA)n microsatellite loci using a streptavidin - magnetic bead enrichment protocol and designed primers for 11 loci. All 11 loci successfully amplified for all 57 kakapo but only one locus was polymorphic and then only for one individual. This individual is the sole surviving kakapo from the now extinct Fiordland population. Cross-species amplifications for a taxonomically diverse range of parrot species were successful, particularly in other Australasian species, and some polymorphism was noted. We tentatively suggest that the limited polymorphism in kakapo is a result of the majority of individuals originating from a single, isolated population from Stewart Island. However, the possibility that low genetic variation is, at least in part, a consequence of the lek mating system (i.e. skewed mating success) and hence a species characteristic, cannot be disregarded.

 
50. A junction fragment from an insertion mutation in the mouse shows homology to the tlm oncogene

K. Parker, G. Gaskell, D. Crisafulli and K.A. Raphael
School of Biological Sciences, University of Sydney, NSW 2006

Mouse mutants generated by insertional mutagenesis of foreign DNA can be a useful tool for the isolation of novel genes affecting embryonic development. The insertion mutation used in this study was found among seven transgenic mouse lines made by microinjection of a sheep metalothionein promoter - human Factor IX cDNA construct into mouse eggs (Choo et al., Nucl. Acids Res. 15: 871-883, 1987). One of these lines carries an embryonic lethal mutation, since approximately 25% of the embryos in heterozygous intercross matings die soon after implantation, on or before 8.5 days of gestation. Retarded embryos have been isolated from the uterus at 6.5 and 7.5 days and characterized from histological sections. Southern blotting was used to make a restriction enzyme map of the transgene and the flanking mouse DNA. An ApaI junction fragment was amplified, using inverse PCR, and the fragment has been cloned and sequenced. A FastA search of the Genbank and EMBL databases revealed homology to the tlm oncogene, an oncogene isolated from mouse T-cell lymphoma DNA (Lane et al., PNAS 81: 2227-2231, 1984), which is frequently altered in mouse T-cell leukemias and lymphomas (Lane and Tobin, Nucl. Acids Res. 18: 3410, 1990). The occurrance of the prenatal lethal phenotype together with disruption of the tlm related sequence strongly suggests that the tlm related gene plays a critical role during early post-implantation development in the mouse. Work is continuing to further elucidate the nature of the mutation and the role of the wild type gene during mouse development.

51. Protecting biotechnology related inventions

Jennifer L. Ross
Davies Collison Cave (Patent and Trade Mark Attorneys), 1 Little Collins Street, Melbourne, Vic 3000

Biotechnology related inventions encompass a wide range of biological processes and components for commercially exploitable outcomes. You can protect your intellectual property rights in your invention by obtaining Patent protection.

A patentable invention is loosely defined as one that is new, not obvious (ie inventive), to people who understand the technology and useful. It is important to emphasise that there is human intervention in biological processes to arrive at a particular outcome and the outcome is generally a commercially useful product such as insulin, a new AIDS vaccine, a new method in molecular biology, a test kit for a disease, different types of wine and beer, a new variety of disease resistant plant, a flower with an unusual colour or a commercially useful transgenic animal.

It is important to note that you should not publicly disclose (ie. publish, publicly discuss or market/sell) your invention before you file for a patent as this will likely invalidate your right to protection.

52. Systematics of Astrotricha

Jenny Saleeba, Kinnie Ho, Kathie Downs, Bruce R. Lyon and Murray Henwood
School of Biological Sciences A12, The University of Sydney, NSW 2006

Astrotricha is a morphologically distinctive genus of endemic Australian Araliaceae (the ivy family). The genus ranges from far north Queensland to the Grampian Range of south-western Victoria with a disjunct species (A. hamptonii) restricted to the Hammersley Range of Western Australia. There are 16 formally recognised species and a further 10 taxa of Astrotricha are recognisable on the basis of a combination of fruit and leaf characters. Within the geographical range of the genus, taxa are distributed in diverse patterns: parapatric, allopatric, sympatric and disjunct. Many of the morphologically distinct taxa appear to hybridise naturally. The use of a limited number of morphological characters, the lack of reproductive isolation and the relatively complex spatial distribution of the putative taxa has hampered not only the utilitarian recognition of taxa, but also prevented the investigation of the infrageneric phylogenetic and biogeographic relationships of this genus. Nucleotide sequence information obtained from eukaryotic nuclear ribosomal RNA genes (rDNA) provides a valuable tool for phylogenetic reconstruction at various taxonomic levels. In the current study, the ITS region of rDNA was chosen to test the intuitive classification of Astrotricha and to investigate the phylogeny of its component taxa. By using consensus oligonucleotide primers, we have amplified the rDNA from various Astrotricha taxa. Following direct sequencing of the PCR product, the DNA sequence data obtained from each taxon were aligned. Preliminary results indicating the utility of this source of molecular data to problems of this type will be presented.

 
53. Molecular genetics of host manipulation and competition in an insect parasitoid host interaction

Markus Beck, Gitta Siekmann, Heidrun Lorenz, Ulrich Theopold and Otto Schmidt
Department of Crop Protection, University of Adelaide, Glen Osmond, SA 5064

Endoparasitoid wasps that develop inside the body cavity of another insect rely on maternal secretions to adjust to antagonistic host conditions. In laboratory and wild populations of the inchneumonid wasp Venturia canescens, one of the ovarian-specific proteins has been found to exist in two allelic forms. Using the allelic difference as a diagnostic marker, two substrains were established, which revealed two distinct egg deposition strategies. One substrain prefers to deposit eggs into non-parasitised caterpillars, whereas the other substrain has the tendency to super-parasitise. In addition, the two substrains display different ovarian phenotypes, which seem to differentially influence egg maturation and egg numbers in the oviduct.
 

54. Genetic variation on islands: Mhc polymorphism in populations of the Australian bush rat

Jennifer Seddon1 and Peter Baverstock2
1Centre for Conservation Genetics, Southern Cross University, Lismore
2Graduate Research College, Southern Cross University, Lismore

The effects of isolation and fragmentation on Major Histocompatability Complex (Mhc) variation have been explored for a series of relict island populations of varying sizes. Relict populations of Rattus fuscipes greyii, found on 14 islands off the South Australian coast, were compared with two mainland populations. The level of variation for 249bp of the second exon of RT1.Ba was screened using Temperature Gradient Gel Electrophoresis (TGGE). Overall genetic variation was high, with 36 alleles identified. A codon deletion was identified in approximately half of the alleles. Despite the influence of balancing selection acting on polymorphic Mhc loci, most smaller island populations were monomorphic. Larger populations retained substantial variation. A lack of geographical or temporal subdivision can be attributed to the retention of ancestral polymorphisms or to structuring in the source population prior to fragmentation.

 
55. Understanding the social organisation of koalas using molecular methods

D.E. Sharkey and W.B. Sherwin
School of Biological Science, University of NSW, Sydney 2052

The social structure of the koala (Phascolarctos cinereus) appears to be organised around a male dominance hierarchy. Ecological data has shown that dominant males move more frequently and have larger home ranges than sub-ordinate males. Consequently they are thought to have a higher mating success because of greater access to mates. Younger males have been found to disperse from their natal areas, whilst females are more likely to be philopatric and breed in their natal areas. The combination of dominance hierarchies and female natal philopatry may result in inbreeding within groups of koalas. Also, male-biased dispersal and female natal philopatry is expected to result in higher relatedness amongst females in a group of koalas than amongst males. In this study, molecular methods have been used to confirm the social organisation of koalas as predicted from ecological studies. Six hypervariable microsatellite loci have been used to analyse 380 koalas from a population in northern NSW. Preliminary results indicate that the structure and dynamics of koala populations is more complex than ecological studies suggest. The study highlights the importance of using both ecological and molecular methods to understand social behaviour in koalas.

56. Sex-specific genes of Bactrocera tryoni

Deborah C.A. Shearman and Marianne Frommer
FFRC, School of Biological Sciences A12, The University of Sydney, NSW 2006

In Drosophila melanogaster, the doublesex (dsx) gene is the last gene in a heirachy of genes that control most aspects of somatic sexual differentiation. dsx is also the first bifunctional gene: both male- and female-specific protein products are produced by differential splicing of the dsx pre-mRNA. Female-specific splicing of the dsx pre-mRNA is activated by the binding of TRA and TRA-2 to a region designated the dsx repeat element (dsxRE), which lies within the non-coding region of exon 4. Male-specific splicing is the default process as a fucntional male-specific DSX is produced in the absence of functional TRA protein. The DSX proteins have a common amino terminus followed by a sex-specific carboxyl terminus and both proteins have been shown to differentially regulate the expression of the yolk protein (yp) genes in the fat body of adult flies. Both the male- and female-specific DSX proteins have been shown to bind to the same target sites within a 127bp enhancer region, which lies in the intergenic region of yp1 and yp2, and repress or enhance transcription respectively.

A homologue of dsx has been shown to be present and expressed in a sex-specific manner in Bactrocera tryoni (Queensland fruit fly). The two cDNA clones isolated from adult B. tryoni show a significant degree of homology at the amino acid level to the female-specific or male-specific cDNA of D. melanogaster dsx. Northern blot analysis confirms that these fragments represent a female-specific and male-specific transcript expressed in the adult. Analysis of the 3' non-coding region of the putative female transcript has identified four 13 nucleotide repeats which are 8/13 bases identical to the tra/tra-2 recognition sites found in exon 4 of dsx in D. melanogaster. This would suggest that homologues of the tra and tra-2 genes also exist in B. tryoni. To investigate whether the function of B. tryoni dsx is the same as that in D. melanogaster, isolation of the yolk protein genes, yp1 and yp2 is underway. We propose that the sex determination pathway in B tryoni and D. melanogaster is the same from the bottom up, despite a difference in the initial sex-determination signal ó the X:A ratio in D. melanogaster and a dominant male-determiner on the Y-chromosome in B. tryoni.

 
57. The CGH (Comparative Genomic Hybridisation) experience of an oncology cytogenetics laboratory

Lisa Robson and Ellie Smith
Department of Cytogenetics, Royal Alexandra Hospital for Children, Westmead

Solid tumours grow poorly in routine tissue culture and thus the area of solid tumour cytogenetics has been relatively underexplored. CGH enables imbalances of the entire genome to be assessed in a single experiment without the need for cultured preparations. Tumour (test) DNA labelled with a green fluorochrome is combined with normal (reference) DNA labelled red. This DNA cocktail is hybridised to metaphase spreads from a normal control. When present equally, green (test) and red (reference) fluorochromes are mixed. Gain/loss of tumour DNA results in increased/decreased green fluorescence.

We received 2 tumour samples - uterine leiomyoma and a neuroendocrine tumour of the small bowel. Extracted DNA was directly labelled by nick translation (VYSIS kit), coprecipitated with differentially labelled reference DNA (VYSIS) and unlabelled COT-1 DNA (GIBCO). Following denaturation of the DNA cocktail and control chromosomal DNA (on metaphase spreads), hybridisation (several days), stringency washes and counterstaining with DAPI (SIGMA) were performed. A fluorescence microscope (ZEISS) with CGH software (Applied Imaging) was used. Adhering to strict criteria for each cell, a minimum of 10 cells is recommended for statistically significant assessment. The results are then presented on a karyotyped intensity ratio profile. Important factors for implementation in a routine laboratory include interpretation of small gains/losses and limit of resolution. CGH is a potentially useful tool in oncology genetics.

 
58. Phylogenetic analysis of Australian drywood termites (Isoptera, Kalotermitidae) based on nucleotide sequence from two mitochondrial DNA genes

Graham J. Thompson and Ross H. Crozier
School of Genetics, La Trobe University, Victoria 3083

Mitochondrial DNA sequence data from genes cytochrome b (CytB) and cytochrome oxidase II (COII) are characterized and analyzed for phylogenetic content among 27 species of termites, particularly Australian lineages of the "drywood" termites (Kalotermitidae). Preliminary analysis indicates that COII is more conserved than CytB, but that both genes are informative in resolving phylogenetic relationships over varying divergence times. The overall nucleotide content of COII was A+T biased (64.7%) and significant compositional heterogeneity existed for two remote sequences. Pairwise sequence divergence estimates reflect the antiquity of termite diversity at the family level, but comparisons within the Kalotermitidae remain above the saturation point for transitions, indicating that the degree of homoplasy in this gene is not likely to confound cladistic topologies from unweighted data sets. Neighbour-Joining and Maximum Parsimony trees are largely congruent, especially above the generic level.

 
59. Multiple representatives of the mariner family of transposable elements in the tephritid fruit fly Bactrocera tryoni

Catherine Turney, John Sved and Marianne Frommer
School of Biological Sciences A12, University of Sydney, NSW, 2006

Members of the mariner family of transposable elements have been found to be abundant in the genome of the Queensland fruit fly, Bactrocera tryoni. Initially, degenerate oligonucleotide primers designed to amino acid sequences conserved amongst mariner element transposases were used to PCR amplify fragments of ~480-490bp from B. tryoni genomic DNA. Following cloning and DNA sequencing of multiple amplified fragments, five distinct types of mariner elements were identified. These grouped with representatives of three major subfamilies of mariner elements during phylogenetic analysis. Using the same PCR-based approach, diverse mariner sequences were also isolated from Bactrocera neohumeralis, a sibling species of

B. tryoni, and an additional tephritid, Bactrocera jarvisi.

Genomic DNA library screening and inverse PCR were used to isolate multiple partial or full-length representatives of two mariner element subfamilies present in the B. tryoni genome. These distinct mariner element types share ~23% amino acid identity in the predicted translations of their transposase genes, including many of the residues that are moderately or highly conserved amongst transposases encoded by mariner family members. However, all B. tryoni copies characterized appear defective, containing multiple mutations in transposase gene regions. The 'mellifera' subfamily elements (=Btmar1 type) appear to be present in a very high copy number within the B. tryoni genome, with library screening yielding an estimate of ~900 Btmar1-containing clones per haploid genome equivalent. In contrast, Southern blot analyses suggest that representatives of the 'irritans' subfamily of mariner elements (=Btmar2 type) have a low copy number in the B. tryoni genome.

60. Transfection of tammar mallaby fibroblasts

M.J. Wakefield and J.A.M. Graves
La Trobe Univertsity

Primary cultures of marsupial fibroblasts have been a valuable tool for the study of marsupial genetics. The limited lifespan of primary cultures has however, restricted their use for many experiments. In order to develop a permanent line of marsupial fibroblasts, we have established a protocol for the transfection of primary marsupial fibroblasts using the Fugene6 reagent (Boehringer Mannheim), utilizing a green fluorescent protein (GFP) expression vector as a control.

 
61. Characterisation of w-globin in a marsupial supports a new model for b-globin gene evolution

David Wheeler1, Rory Hope1, Steven Cooper2, Robert Holland3, Andrew Gooley4 and Morris Goodman5
1Department of Genetics, University of Adelaide, South Australia
2Evolutionary Biology Unit, South Australian Museum
3School of Physiology and Pharmacology, University of New South Wales
4Macquarie University Centre for Analytical Biotechnology, New South Wales
5Department of Anatomy and Cell Biology, Wayne State University, Detroit, Michigan

Previous molecular studies in Sminthopsis crassicaudata and Didelphis virginiana led to the suggestion that extant marsupials have a b-globin cluster consisting of just two genes: e-globin expressed in the neonate, and b-globin expressed in the "adult". Recently, Holland & Gooley (1997) isolated the gene product from a third b-like globin gene, w-globin, in the marsupial Macropus eugenii (Tammar wallaby). The partial AA-chain of the w-globin has more sequence identity with bird b-like globins than mammalian b-like globins. To investigate this observation further, a PCR approach was adopted to isolate and determine the sequence of Tammar w-globin. Phylogenetic analysis based on this sequence showed that w-globin grouped with the bird globins in a manner that can not be accommodated by current models of b-globin evolution. The phylogenetic evidence obtained in this study has been used to develop an alternative model for the evolution of b-globin genes in birds and mammals. A feature of the model is that w-globin was produced by a duplication that occurred in an ancestral b-globin gene that existed before the divergence of the mammals and the birds.

 
62. Evolution of neural development in the arthropods: a molecular approach

G. Whitington and P. Whitington
Division of Molecular and Cellular Biology, Universtiy of New England, Armidale, NSW

Evolutionary changes in the structure of organisms result from modifications to pre-existing developmental programs. We are interested both in how processes of neural development have been altered during evolution and the evolutionary links between the different major groups of arthropods.

The ability to analyse arthropod nervous systems at the level of single identified neurons assists the identification of homologous cells and to thereby recognise conserved and changed features. There is compelling evidence for a strongly conserved program of neural development across the insects. A comparative study of axon growth suggests that this conservation may extend to the malacostracan crustaceans. Myriapods are traditionally regarded as being the group of arthropods most closely related to insects. However an earlier study of embryonic axon growth in the centipede revealed major differences to the insects and crustaceans. This earlier study relied on similarity of cell position and axon morphology to establish homology.

Another approach is to examine expression of orthologous genes in the three arthropod groups. A good choice is the gene even skipped (eve), which shows a highly conserved pattern of expression in a specific subset of embryonic neurons in insects as divergent as Drosophila and the grasshopper. Based on sequence of the highly conserved homeodomain region of Drosophila and mouse, we have cloned eve from crayfish(crustacean) and centipede(myriapod) embryonic cDNA using a nested PCR approach. The sequence has been extended either side of the homeodomain using RACE and expression of eve is being determined via in situ hybridisation using species specific eve riboprobes on fixed embryos.

 
63. Evidence of the retrotranscriptional origin of the Gpdh gene in the honey bee Apis mellifera

Tomasz M. Wilanowski and John B. Gibson.
Molecular Genetics and Evolution Group, Research School of Biological Sciences, The Australian National University, G.P.O. Box 475, Canberra, ACT 2601

Unlike other insects studied up to date, stinger bees (Apis sp.) express only one isozyme of sn-glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8). We have investigated the Gpdh gene in the honey bee (Apis mellifera) and found that it lacks most introns present in other organisms. 5' and 3' RACE analyses showed that the A. mellifera Gpdh gene expresses only one mature transcript. The lack of introns prevents the gene from producing different GPDH isozymes by alternative splicing, which occurs in Drosophila melanogaster and other fruit flies. We suggest that the intron loss arose via reverse transcription of a spliced Gpdh transcript. Because other Apis species display the same GPDH isozymic phenotype as A. mellifera, we believe that the introns were removed from the Gpdh gene prior to the speciation of the Apis genus.

Northern blotting experiments revealed an accumulation of unspliced Gpdh pre-mRNA in A. mellifera, which probably reflects splicing inefficiency. However, it is also possible that splicing is a regulated step in Gpdh expression in the honey bee.

 
64. Molecular evolution in the carnivorous marsupial genus Myoictis

Jodie Young1, Carey Krajewski12, Steve Donnellan3 and Michael Westerman1
1Department of Genetics and Human Variation, La Trobe University, Bundoora, Vic. 3083
2Department of Zoology, Southern Illinois University, Carbondale, Illinois 62901-6501, USA
3South Australian Museum, North Terrace, Adelaide, South Australia 5000

The genus Myoictis is one of four endemic genera of dasyurid marsupials found in Papua New Guinea. Currently two species are recognized - Myoictis melas and Myoictis wallacei - though the former may comprise two subspecies. We have examined DNA sequences from two mitochondrial genes (Cytochrome b and 12S ribosomal RNA) as well as allozyme data from a number of geographically separate populations. The results of our analyses will be presented along with their implications for a revised taxonomy of this genus.