U. Kuhnlein

DNA fingerprinting: a tool for determining genetic distances between strains of poultry

SummaryDNA fingerprinting, a technique based on the detection of hypervariable minisatellite regions in DNA restriction fragments, was tested for its applicability to conduct population genetics in poultry. Using MspI digestion and phage M13 DNA as a probe, between 25 and 35 minisatellite-containing DNA fragments were observed per bird. Comparison of the banding pattern of offspring with their parents revealed that the bands were inherited as stable genetic traits. The variability of the DNA fingerprinting pattern was reduced in inbred strains. DNA fingerprints of chickens from five well-defined populations of known genetic relationships were analyzed and indices of genetic distances were computed. They correctly reflected the history of these strains, indicating that DNA fingerprinting may be a powerful tool to characterize genetic relationships between different breeding populations of the same species.

Male reproductive tactics in the threespine stickleback– an evaluation by DNA fingerprinting

A random sample of 17 threespine stickleback nests was analysed using DNA fingerprinting. DNA from the guardian male and a random subsample of 10 fry per nest was probed with pYNZ132, a human single–locus VNTR probe which detects a multilocus fingerprint pattern in sticklebacks. Band–sharing indices (BSIs, the proportion of bands shared by two individuals) between the guardian male and its fry were calculated. In 147 of a total of 170 pair–wise comparisons the BSIs varied between 0.40 and 0.77. The guardian male was thought to be the true father of all these fry (p < 0.10). For the remaining 23 fry the BSIs varied between 0.09 and 0.34, suggesting that these fry were fathered by a different male (P<0.06). Once the paternal bands in each legitimate fry were determined, the remaining (i.e. maternal) bands among these fry were compared. Based on the BSIs obtained, the minimum number of females that spawned per nest was determined, and the maternal DNA fingerprints of the legitimate fry were traced back. In one nest five eggs of the sample had been fertilized by a sneaker, in two nests the guardian male had stolen eggs from a rival male, and in another nest one of the eggs was fertilized by a sneaker and three were stolen eggs.

Characterization of hypervariable microsatellite loci in the threespine stickleback Gasterosteus aculeatus

Single locus minisatellite and microsatellite DNA fingerprinting have become increasingly important to studies of relatedness in wild animals (Pembertonet al. 1991, and references therein). Here we describe the characterisation of polymorphic single-locus microsatellites for a fish species the threespine stickleback Gasterosteus aculeatus L. and briefly discuss their potential applicability in studies of the evolutionary ecology of this fish. A total of five repetitive GT/AC clones were sequenced. Using the polymerase chain reaction (PCR) we directly amplified microsatellite loci using the flanking sequences as primers. The primer sequences are shown in Table 1. The number of repeats per locus ranged from 20 to 38. All clones had perfect repeats of the dinucleotide with the exception of one clone which had a transversion of a G to a C near the middle of the repetitive sequence. PCR reactions were performed in 10 ml using the thermostable Taq polymerase under the recommended conditions of the supplier (Pharmacia). About 200 ng of genomic DNA were used per reaction (template DNA was prepared as described in Ricoet al. 1992). Final buffer concentrations were 5 0 m ~ KCI, 10-mM Tris-HCI (pH 8.3 at 21”C), 1 .5-m~ MgCl,, 0.01 % gelatine, 5% deionized formamide, and 100 pmol of each primer. DNA was denatured at 94°C prior to the addition of 0.5 units of Taq polymerase and a mixture of four deoxynucleotides. The final concentration of each iiucleotide was 125 pmol and 4.5 pmol of [CX-~~SI~ATP were included. Subsequently, the temperature was cycled between 92°C for 60 s, 58°C for 60 s and 72°C for 120 s for 35 cycles using a DNA Thermal Cycler (Perkin Elmer Cetus Corp.). The reaction products were resolved by electrophoresis in an 8% denaturing polyacrylamide sequencing gel (Sambrook et al. 1989) where single base length differences could be resolved. A sequencing ladder was used as a molecular weight marker. Using genomic DNA from nine randomly collected, and therefore presumably unrelated individuals, we esti-

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal.

Incidence of endogenous viral genes in two strains of white leghorn chickens selected for egg production and susceptibility or resistance to Marek's disease.

SummaryEndogenous viral (ev) genes related to the avian leukosis virus were classified in two differentially selected strains of Leghorns in order to investigate whether such genes affect production traits. Strain K had been selected for resistance to Mareks disease (MD) and for high egg production and egg weight, whereas strain S had been selected only for MD susceptibility. Except that founders of strain K included a few commercial birds, both strains were derived from a common genetic base. DNA restriction fragment length analyses of 110 strain K and 94 strain S birds revealed the presence of 8 different ev-genes, 6 of which were identical to previously identified loci. This result was confirmed by assays for group specific antigen (gs-antigen), the product of the gag region of the ev-genes. The levels of gs-antigen in the birds closely followed what had been predicted from data obtained from previously described ev-genes. Both strains had a similar average number of ev-genes per bird (3.5 and 3.2 for strains S and K, respectively). However, strain K carried only five different ev-genes while strain S carried seven. Four of these loci were present in both strains. Among the ev-genes absent or occurring less frequently in strain K were those that code either for infectious endogenous virus (ev-10 and possibly ev-19) or for the internal viral gag-proteins (ev-3). Only those ev-genes which are transcriptionally silent or which code for the viral envelope gene were present in increased frequencies in strain K. The results indicate that selection for egg traits and/or Mareks disease resistance reduces the frequency of ev-genes which produce endogenous virus or the viral gag-proteins.

Identification of growth hormone DNA polymorphisms which respond to divergent selection for abdominal fat content in chickens

SummaryTwo strains of meat-type chickens which had been derived from the same genetic base, but were selected for high or low abdominal fat content, respectively, were analyzed for polymorphisms in the growth hormone gene (GH). A total of four DNA polymorphisms were identified, one at a SacI restriction site and three at MspI restriction sites. Restriction mapping indicated that all polymorphisms were in exons and/or introns and not in flanking regions of the gene. The incidence of GH polymorphisms was determined in 20 chickens from each strain and significant differences were observed for two of the four polymorphisms. Analysis by DNA fingerprinting using (CAC)5 as a probe indicated that the inbreeding coefficient was 0.1 in both strains and that random genetic drift was minimal. Thus, the selection for abdominal fat appears to have affected the frequency of alleles of the growth hormone gene. Whether this is the direct consequence of an altered growth hormone gene on fat metabolism or reflects linkage to an allele of a neighbouring gene remains to be determined.

Development and validation of a homologous radioimmunoassay using a biologically active recombinant turkey prolactin

1. A new homologous radioimmunoassay has been developed for the measurement of turkey prolactin. 2. A 25000 kDa purified recombinant derived turkey prolactin (rtPRL), the biological activity of which was tested using a crop sac assay, was used as immunogen for the production of rabbit antiserum. In this biological test, the rtPRL was as active as the ovinePRL. 3. The radioligand (rtPRL) was labelled with 125I and the assay allowed the detection of standard doses of rtPRL ranging from 400 pg/tube to 50 ng/tube. 4. No cross reaction with chicken luteinising hormone and recombinant chicken growth hormone was detected. 5. The within and between assay coefficients of variability were 5.0 +/- 2.7% and 16.3%, respectively. The overall mean recovery ratio was 1.01. 6. The dose-response curves obtained with serial dilution of plasma and pituitary from turkey hens at different physiological stages and from male turkeys were parallel to those obtained with standard rtPRL. 7. The measured concentration of prolactin was 5 times higher in plasma from incubating than laying turkey hens, and the pituitaries from incubating hens contained 2 and 4 times more prolactin than those of laying and out of lay hens or males, respectively. 8. To further assess the validity of the assay, we measured changes in plasma concentration of prolactin in turkeys following stimulation with chicken vasointestinal peptide (cVIP). A single injection of 1 or 10 micrograms/kg body weight of cVIP to laying hens produced a large and rapid increase in plasma prolactin. 9. This new radioimmunoassay appears to be high for the measurement of turkey prolactin.

Production and characterization of recombinant turkey prolactin

1. Recombinant turkey prolactin (rctPRL) was produced as a fusion protein in E. coli, purified by affinity chromatography followed by cleavage with thrombin. The final yield of the released rctPRL (> 90% purity) was 1-2 mg/l of bacterial culture. 2. Recombinant tPRL co-migrated with the main immunoreactive band (25 kDa) in turkey pituitary extracts and was identical to natural tPRL except for the addition of three amino acids (Gly-Ser-Ser) resulting from the cloning strategy at the amino terminal end. 3. The bioactivity of the rctPRL was equipotent to ovine PRL in a rabbit mammary explant system and in the Nb2 lymphoma mitogenic assay.

Identification of Markers Associated with Quantitative Trait Loci in Chickens by DNA Fingerprinting

Three approaches for identifying VNTR alleles associated with quantitative traits in chickens are described. One approach is based on the comparison of well-defined selected and non-selected control strains. The second approach is based on analyzing chickens within a breeding population ranked according to specific traits and the third approach involves segregation analysis. In this latter approach a large number of offspring of a single male segregating for a quantitative trait are produced and tested for trait association of the male DNA fingerprinting bands. In all cases pooled DNA samples of birds, rather than individual samples, are analyzed and band intensity is assumed to reflect the relative frequency of an allele. Examples from the literature and from our laboratory indicate that these methods permit the identification of DNA fingerprinting bands associated with quantitative traits. After developing locus-specific probes for these bands it should ultimately be possible to detect and map quantitative trait loci.

Synergism between the endogenous viral loci ev6 and ev9 in inducing immunological tolerance to avian leukosis virus.

1. The course of infection by exogenous avian leukosis virus was followed in a commercial strain of White Leghorn domestic fowls by measuring viral antigen in feather pulp and egg albumin. Ten days after hatching, 2 out of 360 birds tested positive and at 286 days of age about 60% of the birds had been antigen positive at least once. 2. Among the antigen positive birds, two groups could be distinguished: those which permanently and those which transiently expressed viral antigen. Permanent antigen expression was associated with low antibody titres, while transient antigen expression was associated with high antibody titres. 3. The strain segregated for the two endogenous viral genes ev6 and ev9, both of which express endogenous viral envelope protein, and have been implicated in affecting immune-responsiveness. The antibody titre in individuals positive for both ev6 and ev9, was significantly lower than in those which had none or only one of the two ev-genes. In addition, individuals positive for both ev-genes occurred more frequently in the group permanently positive for viral antigen than in the group transiently antigen positive. 4. The results indicate that there was a strong synergism between ev6 and ev9 in reducing the antibody response to exogenous avian leukosis virus infection, perhaps by inducing immune tolerance or interfering with antibody formation.