Françoise Coudert

At Least One Class I Gene in Restriction Fragment Pattern-Y (Rfp-Y), the Second MHC Gene Cluster in the Chicken, Is Transcribed, Polymorphic, and Shows Divergent Specialization in Antigen Binding Region

MHC genes in the chicken are arranged into two genetically independent clusters located on the same chromosome. These are the classical B system and restriction fragment pattern-Y (Rfp-Y), a second cluster of MHC genes identified recently through DNA hybridization. Because small numbers of MHC class I and class II genes are present in both B and Rfp-Y, the two clusters might be the result of duplication of an entire chromosomal segment. We subcloned, sequenced, and analyzed the expression of two class I loci mapping to Rfp-Y to determine whether Rfp-Y should be considered either as a second, classical MHC or as a region containing specialized MHC-like genes, such as class Ib genes. The Rfp-Y genes are highly similar to each other (93%) and to classical class Ia genes (73% with chicken B class I; 49% with HLA-A). One locus is disrupted and unexpressed. The other, YFV, is widely transcribed and polymorphic. Mature YFV protein associated with β2m arrives on the surface of chicken B (RP9) lymphoma cells expressing YFV as an epitope-tagged transgene. Substitutions in the YFV Ag-binding region (ABR) occur at four of the eight highly conserved residues that are essential for binding of peptide-Ag in the class Ia molecules. Therefore, it is unlikely that Ag is bound in the YFV ABR in the manner typical of class Ia molecules. This ABR specialization indicates that even though YFV is polymorphic and widely transcribed, it is, in fact, a class Ib gene, and Rfp-Y is a region containing MHC genes of specialized function.

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeor selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%.Neor andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Intratesticular Inoculation of Avian Leukosis Virus (ALV) in Chickens-Production of Neutralizing Antibodies and Lack of Virus Shedding into Semen

In order to investigate the possibility of producing transgenic chickens by injection of avian leukosis virus-based vectors into testis, we have analyzed the infection rate of testicular cells following inoculation of Rous-associated virus type 1 (RAV-1) into the gonads of adult and 1-wk-old brown leghorn males. Viroproduction, neutralizing antibody production, and vital DNA presence in testis, blood, muscle, and semen were analyzed at various times after infection. Inoculation of RAV-1 into the gonads of adult males resulted in a low level of viroproduction in testis and blood, followed by the appearance of neutralizing antibody 2 or 3 wk later. Neither viroproduction in semen nor viral DNA presence in sperm were detected even though the infected chickens were found to produce RAV-1 in testis. One week after intratesticular inoculation of 1-wk-old males with RAV-1, a high level of viroproduction was found in blood and testis, and viral DNA was detected in gonadal cells. Further, by 6 wk after inoculation, the production of virus decreased in all tissues, viral DNA could not longer be detected in the testis, and neutralizing antibodies appeared in blood. All together these data show that it is possible to infect testicular cells by direct inoculation of RAV-1 in the testis, and that the immune response of both adult and young chickens seems to reduce this infection. Moreover, no evidence of spermatozoa infection was found; this result suggests that RAV-1 inoculation into testis may not induce genetic transmission of virus, and consequently would not be useful in the production of transgenic chickens.

Are chicken Rfp-Y class I genes classical or non-classical?

Mhc genes in the chicken and at least some other gallinaceous birds are organized into genetically independent clusters containing both class I and class II genes. The genes within these clusters are of interest in that they may provide insights into the function and evolution of the Mhc not found previously in the mammalian paradigms. In the chicken the two gene clusters called B and Rfp-Y reside on the same chromosome but are separated by a region supporting highly frequent meiotic recombination such that B and Rfp-Y haplotypes are inherited independently. Genes within Rfp-Y are apparently transcriptionally active but whether any class I molecules encoded within Rfp-Y present peptides in the fashion of classical class I molecules or perform more specialized functions has not been determined. Consistent with the capacity to hybridize under highly stringent conditions with B system class I DNA probes, the Rfp-Y class I loci are about 73% identical in coding region sequence with the classical class I genes located in the B system. At least one Rfp-Y class I locus is transcribed in nearly all organs and exhibits allelic sequence diversity similar to that of classical class I genes. These similarities suggest that the Rfp-Y class I molecules could be functionally nearly identical to their classical B system counterparts. However, distinctive substitutions of residues that are highly conserved in the peptide binding groove of classical molecules occur in the Rfp-Y encoded molecules making it unlikely that they are capable of presenting antigen in the manner of classical class I molecules. This observation together with those made previously for Rfp-Y class II loci demonstrate that the Rfp-Y gene cluster likely represents specialization not previously encountered in the study of Mhc evolution.

Analysis of B complex polymorphism in Rous sarcoma progressor and regressor chickens with B-G, B-F, and B-L beta probes.

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.

Intramagnal insemination of hens can eliminate negative influence of lipofectin on fertilising ability of spermatozoa

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.

Protein Concentrations and Immunosuppressive Properties of Serum in Chickens Experimentally Infected with Avian Tumor Viruses

Sera from chickens affected by Mareks disease or developing Rous sarcoma were investigated. There were changes in the protein fractions, and the amount of alpha and beta fractions was consistently increased. At the same time, immunosuppressive factors were found to inhibit the number of plaque-forming cells in the spleen of mice immunized with sheep red blood cells.

Pathogenicity of a Marek's disease virus for chickens vaccinated with graded doses of herpesvirus of turkeys.

Six different groups of one-day-old chickens were vaccinated with herpesvirus of turkeys (HVT) vaccine at doses of 0, 4, 16, 64, 256 and 1,024 plaqueforming units. At 9 days of age they were challenged with an acute strain of Mareks disease herpesvirus (MDHV). Gross and microscopic lesions in nerves and gonads were investigated and combined for quantitative expression of MDHV pathogenicity. Including the microscopic lesions improved the accuracy of the HVT doses/MDHV response. The qualitative aspects of late microscopic lesions could be related to acquired resistance or to persistence of susceptibility in vaccinated chickens.