Selection for disease resistance related traits is a tool of choice for evidencing and exploring genetic variability and studying underlying resistance mechanisms. In this framework, chickens originating from a base population, homozygote for the B19 major histocompatibility complex (MHC) were divergently selected for either progression or regression of tumors induced at 4 weeks of age by a SR-D strain of Rous sarcoma virus (RSV). The first generation of selection was based on a progeny test and subsequent selections were performed on full-sibs. Data of 18 generations including a total of 2010 birds measured were analyzed for the tumor profile index (TPI), a synthetic criterion of resistance derived from recording the volume of the tumors and mortality. Response to selection and heritability of TPI were estimated using a restricted maximum likelihood method with an animal model. Significant progress was shown in both directions: the lines differing significantly for TPI and mortality becoming null in the "regressor" line. Heritability of TPI was estimated as 0.49 +/- 0.05 and 0.53 +/- 0.06 within the progressor and regressor lines respectively, and 0.46 +/- 0.03 when estimated over lines. Preliminary results showed within the progressor line a possible association between one Rfp-Y type and the growth of tumors.

White Leghorn chicks homozygous for B19 MHC haplotype were selected for 18 generations on tumor regression after inoculation in the wing web with an SR-D strain of Rous sarcoma virus (RSV) at 4 wk of age. Each chick was assigned a tumor profile index (TPI) based on age at death and size of the tumor. During 18 generations, 2,010 birds were divergently selected on TPI for either progression or regression of the tumor (P and R lines). A Brody growth curve was fitted for each bird. Brody function parameters included the asymptotic tumor volume (A), the factor for increased growth in progression phase (K1), the factor for decreased growth in regression phase (K2), age at maximum volume (Tmax), and maximum volume of the tumor (Vmax). Tumor growth curves were found to be different according to line, sex, and restriction fragment pattern Y complex Rfp-Y MHC haplotype (Yw*15, Yw*16, and Yw*17). Within the P line, birds from the Yw*16 haplotype reached Vmax at an earlier age than Yw*15 and Yw*17, but with a lower Vmax value. Within the R line, tumor growth curves of birds from Yw*16 and Yw*17 haplotypes were similar. Rank correlations between the different parameters and TPI were low (between -0.26 and 0.36). Heritability estimated by the sire component was high for Vmax (0.73). Heritabilities of Tmax and K2 were moderate (0.20 to 0.23 for Tmax and 0.18 to 0.21 for K2) allowing these traits to be used as selection criteria. Heritabilities of A and K1 were lower than 0.12. Modeling the growth curve should contribute to better distinction between progressors and regressors.

The coding region of the preproinsulin gene has been cloned and partly sequenced in a variety of marine and terrestrial birds (28 species). All genes showed the "ancestral" structure with a large intron-2. The size of intron-2 changed considerably during the evolution of birds (2.4-4.2kb). The hydrophobicity of signal peptides was conserved. Bird C-peptides were predicted to be 28 amino acids long, but circulating C-peptides would be only 26 amino acids long, with Passer as a possible exception. Bird C-peptides were found to lack the sequences identified in mammals as responsible for peptide bioactivity and the structure of the central part. In contrast, predicted insulin sequences were highly conserved. Only two types of analog were identified: the hypoactive form (GluA8), present only in Anseriformes and the hyperactive form (His A8), present in all other species. Based on 3'-nucleotide sequence analysis (extending into intron-2), birds appeared to be monophyletic. Five groups were clearly identified: Paleognathae, Galliformes, Anseriformes, Passeriformes, and Charadriiformes. Paleognathae were suggested as the basal group, supporting the traditional view of avian evolution. Subsequent branching identified a gallo-anserae group and a group containing all other Neognathae. Surprisingly, Columba livia (Columbiforme order) clustered with Galliformes. With represented species, Procellariiformes and possibly Ciconiiformes, and Pelicaniformes were suggested as paraphyletic, in agreement with conclusions from some studies based on mitochondrial DNA sequences.

AIMS:Lymphoblastoid cell lines derived from Marek's disease virus (MDV) induced tumours have served as models of MDV latency and transformation. They are stable and can be cultured with no detectable MDV genomic alterations upon repeated passaging. An MDV transformed lymphoblastoid T cell line (T9 cell line) has been reported to contain a disrupted MDV BamHI-H fragment and a Rous associated virus insertional activation of the c-myb protooncogene. In an attempt to define the respective participation of c-myb and MDV in the transformed phenotype of T9 cells, an analysis of MDV oncogenic sequences (BamHI-H, BamHI-A, and EcoQ fragments) was performed in these cells.METHODS:Using two different passages of the T9 cell line (late and early passages), the organisation of the MDV oncogenic regions and their expression in these cells were analysed. In vivo assessment of the oncogenicity of the virus contained within these cells was assessed by injecting them into 1 day old chickens.RESULTS:In T9 cells maintained in culture for up to six months (late T9), the MDV ICP4 gene was disrupted, whereas the meq gene was actively transcribed. The alterations of the MDV genome in these cells correlated with the inability of the virus to induce the classic signs of Marek's disease in 1 day old chickens. However, early T9 cells submitted to a limited number of passages induced classic MDV pathogenicity, as efficiently as the MDV control cell line (T5), and did not show gross structural changes in the oncogenic MDV sequences.CONCLUSIONS:Although the expression pattern of the MDV oncogenes in early T9 cells was identical to the one reported for other MDV transformed cells, longterm culture of an MDV transformed cell line containing a RAV insertional activation of the c-myb protooncogene led to the disruption of the MDV BamHI-H and BamHI-A oncogenic regions. In the late T9 cells MEQ was the only detected MDV oncoprotein. These results suggest that in the late T9 cells the truncated MYB protein compensates for the loss of MDV oncoproteins and reinforce the possibility that MEQ and MYB cooperate in the maintenance of the transformed state and the tumorigenic potential of these cells.

Only some of the gene transfer techniques can be used as tools for insertion of foreign DNA into the cells of chicken testes. In our experiments a possible transfer of lipofectamine plasmid (linearized plasmid DNA-pGL3 H CMV luc) mixture via direct injection into the testes of the young cocks was verified. In Group 1, testes of 17 cocks were inoculated at the age of 2 weeks, in Group 2, testes of 21 cocks were inoculated at the age of 4 weeks. Twelve days after the inoculation a test for the presence of the inserted plasmid was performed. All analyzed samples except one in Group 2 were found highly positive, by PCR. No positive signals were found with DNA isolated from the testes 6 weeks after inoculation of the testis of both groups nor in the semen of the cocks allowed to grow to adulthood. The transferred plasmid complex has nor, been integrated into the genomic DNA.

The production of chicken chimeras using donor and acceptor cells which can be of opposite sex has necessitated the utilization of methods developed to distinguish the sex of chickens. We demonstrate one of these methods, based on the polymerase chain reaction which amplifies the EcoRI repeat unit of the fowl W chromosome, and how this technique may be used to sex various cell types in chickens as well as small numbers of blastodermal cells. Our results demonstrate the ability to sex chickens using EcoRI primers, specific for the W chromosome, from as little as 2 ng of female genomic DNA isolated from blood and feathers--the latter being the result of DNA extraction from only one feather. Also evident in this study is the detection of the W chromosome by PCR from approximately 50 blastodermal cells originating from the developing blastodisc at stage X.

1. After intramagnal insemination egg production decreased drastically during the first two days and was equivalent to egg production of hens inseminated intravaginally for the remaining period of collection. 2. After magnal insemination, the fertility of eggs collected during the first week was 36.2% and only 3.6% during the second week. 3. In the case of intramagnal insemination, egg fertility in the first week was 88.1%, in the second week 81.8% and the third week 52.3%. 4. The eggs laid during the first day after intramagnal insemination were 83.3% fertile, indicating that treated spermatozoa fertilised the newly ovulated egg within 20 minutes of ovulation.

Stage X blastodermal cells were isolated from freshly laid unincubated Brown Leghorn chicken eggs. Five hundred cells from Stage X Brown Leghorn embryos were injected into the subgerminal cavity of White Leghorn unincubated embryos exposed to 550 rad of gamma irradiation from a cesium-137 source. Of 712 White Leghorn embryos that were irradiated and injected with Brown Leghorn blastodermal cells, 52 (7.3%) survived to hatching. Somatic chimerism was examined in the melanocyte population and erythroid lineage. The presence of brown feathers indicating donor cell contribution to melanocyte pigmentation was observed in 23 (44%) out of the 52 hatched chicks. Analysis of blood DNA was performed using a probe that revealed an endogenous retroviral gag fragment specific for the donor genome. Three out of these 23 chimeric chickens exhibited the gag-specific fragment. To test germline chimerism, chickens that reached sexual maturity were mated with Brown Leghorns. Three somatically chimeric hens produced Brown Leghorn progeny at a rate of 30.7, 9.2, and 2.9% respectively, thus proving donor cell contribution to the germline differentiation. Chimeric chickens obtained after injection of nonirradiated embryos exhibited a lower extent of chimerism at the feather level and did not show any chimerism in the erythroid lineage and the germline, thus demonstrating the value of the use of compromised recipient embryos to produce chimeras in chickens. Nevertheless, the extent of somatic chimerism could not be used to predict the germline chimerism.

Molecular polymorphism of the B complex was studied in serologically defined B19 haplotypes by use of class I, class II, and class IV probes in Southern blot experiments in chickens. All chickens studied shared identical class IV restriction patterns. In contrast, class I and class II probes revealed six and five subtypes of B19 haplotype, respectively. These subtypes may be resolved in three homozygous genotypes and their corresponding heterozygous combinations. Previous genetic selection allowed us to distinguish two subpopulations in these B19 chickens with regard to the fate of Rous sarcoma virus (RSV)-induced tumors. Molecular genotyping was applied to B19 chickens challenged with RSV in order to determine whether there is a correlation between one of the molecularly defined subtypes and the progressor/regressor phenotypes of the chickens. None of the molecularly defined subtypes correlated with the progressor or regressor phenotype of the challenged birds.