Use of ev loci as a measure of inbreeding in domestic fowls.

1. The endogenous avian leukosis virus (ev) loci present in 9 lines of domestic fowls have been partially characterised and the average heterozygosity of the loci in each line calculated. 2. Using these data an estimate of the coefficient of inbreeding of the lines was derived; this estimate of the extent of inbreeding is compared with the mating history of the lines. 3. This method provides the first means of directly assessing the degree of inbreeding of fowl lines: assumptions implicit in the method are discussed.

Genetic transformation of chickens using irradiated male gametes.

Results have been obtained which corroborate those of Pandey and Patchell (Molec. Gen. Genet., 186, 305, 1982) in demonstrating that genetic material from irradiated semen is incorporated into the embryo and expressed, albeit at rather a low rate, and is subsequently transmitted to progeny of the transfected birds. The method provides a technically straightforward means of transferring genetic material where rapid and reliable means of detecting the transferred gene exist. An advantage of the method is that regulatory regions are likely to be carried with the transferred gene but there is equally a disadvantage in the simultaneous transfer of unwanted material.

Isolation of lambda amp3 genomic recombinants coding for antigens of Eimeria tenella.

Eimeria are the causative agents of coccidosis, a disease which is of world wide economic importance in the poultry industry. Immunity resulting from infection is species-specific and both antibody and cell-mediated responses have been implicated. As an initial step in the development of a genetically-engineered vaccine against coccidiosis, libraries of EcoRI-digested genomic DNA from E. tenella have been constructed in Escherichia coli using the expression vector lambda amp3. Screening of the libraries with serum from chickens immunized by infection has identified at least 24 different recombinant phage which produce eimeria antigens fused to beta-galactosidase. A significant proportion of the Eimeria DNA inserts cross-hybridise with each other and contain sequences which are highly represented in the genome. The identification of these clones will enable the isolation of intact genes from E. tenella DNA and facilitate detailed analysis of the antigens and immune responses.

Functional analysis of reverse transcription by a frameshift pol mutant of murine leukemia virus.

Endogenous reverse transcription by wild-type murine leukemia virus (MuLV) was compared to that catalyzed by clone 23, a pol mutant containing a reverse transcriptase protein which lacks the carboxyl-terminal third of the molecule (J. G. Levin, S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin (1984), J. Virol. 51, 470-478). Competition immunoassays revealed that mutant virions contain normal amounts of polymerase protein, indicating that the lack of carboxyl-terminal sequences does not alter normal processing of enzyme precursors. Although the mutant enzyme was previously shown to have the ability to copy and degrade RNA:DNA hybrids, the present study demonstrates that it is defective in functions required to generate full-length copies of viral DNA. Analysis of products of endogenous reverse transcription showed that minus-strand strong-stop DNA is formed and that mutant virions synthesize a series of minus-strand DNA intermediates up to 2.2 kb in length. Comparison of mutant and wild-type MuLV reaction products indicated that the 2.2-kb termination site of the mutant corresponds to a normal pausing region for the wild-type enzyme. Computer analysis of sequences and structure within pausing regions suggested the involvement of C-rich consensus sequences plus multibranch loop structures in the general phenomenon of enzyme-pausing during reverse transcription.

Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure.

The molecular defect in the nonconditional B-tropic MuLV pol mutant, clone 23 (Gerwin et al., J. Virol. 31:741-751, 1979), has been characterized by recombinant DNA technology. The entire mutant genome was cloned from an EcoRI digest of integrated cellular DNA into bacteriophage lambda Charon 4A and then subcloned at the EcoRI site of pBR322. NIH-3T3 cells transfected with the plasmid clone, termed pRTM (RTM, reverse transcriptase mutant), reproduced the properties of clone 23 virus-infected cells. In vivo ligation experiments involving cotransfection of subclones of pRTM and wild-type murine leukemia virus localized the defect in the clone 23 genome to an approximately 400-base-pair region in the pol gene between the SalI and XhoI sites. Sequence analysis of this region in the wild-type and mutant genomes revealed that the mutant has one additional C residue located 231 bases downstream of the last base of the SalI recognition site. This 1-base insertion brings three TGA termination codons into phase. Thus, the mutation in clone 23 leads to premature termination of translation, explaining the presence in clone 23 virions of a truncated polymerase with low levels of enzymatic activity. It was previously shown that the gag precursor is cleaved normally in clone 23-infected cells; therefore, if a virus-coded protease is involved in this cleavage, it must be encoded by sequences upstream of the reverse transcriptase region of the pol gene. This consideration, coupled with the observed molecular weight of the mutant polymerase and our precise determination of its C terminus, have led to a proposal for the genetic organization of the murine leukemia virus pol gene.

Metabolism of viral RNA in murine leukemia virus-infected cells; evidence for differential stability of viral message and virion precursor RNA.

Molecular hybridization techniques were used to examine the stability of viral message and virion precursor RNA in murine leukemia virus-infected cells treated with actinomycin D. Under the conditions used, viral RNA synthesis was inhibited, but viral protein synthesis continued, and the cells produced noninfectious particles (actinomycin D virions) lacking genomic RNA (J. G. Levin and M. J. Rosenak, Proc. Natl. Acad. Sci. U.S.A. 73:1154-1158, 1976). Analysis of total RNA in virions revealed that the amount of hybridizable viral RNA decreased steadily after the addition of actinomycin D and by 8 h was 10% of the control value. Studies on fractionated viral RNA showed that this low level of hybridization is due to residual 70S RNA in the virion population. The results indicated that viral RNA which is destined to be encapsidated into virions has a half-life of approximately 3 to 4 h. In contrast, other intracellular virus-specific RNA molecules appeared to be quite stable and persisted for a long period of time, with a half-life of at least 12 h. These observations support the idea that two independent functional pools of 35S viral RNA exist within the infected cell: one serving as message and the other as precursor to virion RNA. The existence of two viral RNA pools was further documented by the finding that 12 h after the addition of actinomycin D, when virion precursor RNA was depleted, 35S and 21S viral nRNA species could be identified in polyribosomal RNA as well as in total polyadenylated cell RNA. Surprisingly, 35S and mRNA declined more rapidly than did 21S mRNA, which appeared to be increased in amount.

Complementation with ts mutants of herpes simplex virus.

Tests to detect complementation between ts mutants of herpes simplex virus types 1 and 2 infectious centre and yield of virus assay were investigated. Progeny analysis of both intratypic and intertypic complementation showed a considerable proportion of recombinant or ts+ virus in the progeny; this was more marked in the infectious centre tests. Virus of intermediate temperature-sensitivity was produced in intratypic as well as intertypic complementation. Reduction in the input multiplicity of one of the two mutants in the test to extremely low levels did not prevent complementation, suggesting that non-infectious particles probably contribute to complementation. Demonstration of virus DNA synthesis in mixedly-infected cells at the non-permissive temperature was used to detect complementation between DNA-negative mutants.