In recent years, studies of domesticated animals have demonstrated conclusively that single [ 1,2[ or small numbers of Vf segments [3-51 can be diversified through gene conversion or hypermutation mechanisms 161, to form a fullyfunctional immune repertoire. This offers an alternative to the paradigm derived from human and murine immunology, that the humoral repertoire is founded upon numerous V genes families, each comprising many functional segments of diverse iiucleotide sequence. In this study, we focused on bovine immunoglobulin heavy chains and the mechanisms which diversify germline VH segments present in the into a functional repertoire.As in previous work (7.81, we used the PCR with oligonucleotide primers directed against the heavy chain constant region to amplify VH sequences from bovine splenic cDNA. The products were cloned and sequenced at random. ' Ten complete VH sequences have now been submitted to GenBank to complement those published in our earlier report (81 (accession numbers U55195-55204). Alignment of the complete dataset and pairwise comparison showed that no two sequences were identical but that all cDNAs were derived from a single VH gene family which appeared to be a homologue of human VHII.The high level of conservation of nucleotides encoding the leader and FR3 enabled us to recover members of this gene family from liver genomic DNA with a single set of oligonucleotide primers. Sequencing suggested that the family did not comprise a great many members since several identical clones were isolated at random. This conclusion was supported with data from Southern blots of liver genomic DNA probed with a cDNA clone which showed a very limited number of hybridising fragments. The sequences of 12 unique germline segments have been deposited at GenBank (accession numbers U55164-55 175). The minimum level of homology in pairwise comparison of these segments was >90% indicating that the gene family comprised near-identical members. It was noteworthy that sequence differences between the VH segments in the regions encoding the CDRs were indicative of subgroupings within the gene family rather than differences in potential antigenic specificity. Thus, it appeared most unlikely that cattle could generate a diverse heavy chain repertoire through combinatorial mechanisms alone.We have attempted to elucidate the mechanism of diversification by comparing the germline and cDNA sequences gathered during our investigations. Nucleotide substitutions were preferentially targeted to the CDRs at high frequency resulting in amino acid replacement with greater frequency than substitutions in the leader or FRs. Our analysis produced clear evidence of strand selection in the diversification process, it was evident that purine residues were particularly susceptible to substitution and transitions predominated over transversions. These features were judged to be characteristic of diversification through a process of somatic hypermutation [9, lo]. The sequence information which we gathered also showed that there was...
The herpes simplex virus type 1 deletion variant 1703 apparently fails to synthesize the essential IE2 gene product Vmw63 despite the deletion leaving the gene intact. Sequence analysis revealed that the deletion removes a region to the right of IE2 comprising the 3' end of IE1, UL56 and the 3' part of UL55, stopping 555 bp downstream of the IE2 polyadenylation signal. Further DNA sequencing has shown that there is no secondary mutation in the IE2 gene. Western blot analysis demonstrated that Vmw63 is made at reduced levels compared to that produced by the wild-type virus during immediate early conditions of infection. S1 nuclease protection mapping has revealed that this reduction is also apparent at the level of mRNA synthesis. A direct link between the deletion and the change in mRNA synthesis was provided by the insertion of a deletion-spanning fragment from 1703 into a 17 + genome, which resulted in the recombinant having a 1703-like phenotype. Evidence that down-regulation of IE2 mRNA during immediate early conditions of infection could be due to antisense RNA initiating from the IE1 promoter was obtained by the insertion of a novel transcriptional termination signal between IE1 and IE2 in the variant and the subsequent detection of wild-type levels of IE2 mRNA and protein. IntroductionHerpes simplex virus type 1 (HSV-1) has five immediate early genes (IE1, 2, 3, 4 and 5) encoding the polypeptides Vmwll0, 63, 175, 68 and 12 respectively. Immediate early genes are unique in that they are transcribed in the absence of prior viral protein synthesis and their products are required for the expression of early and late viral polypeptides. The immediate early genes and their encoded proteins have been the subject of close scrutiny and much information has been gained concerning their regulatory activities and functional domains. The isolation and characterization of temperature-sensitive (ts) IE2 mutants demonstrated that Vrnw63 played an essential role in virus replication (Sacks et al., 1985) and the characterization of null mutants in this gene demonstrated the role of Vmw63 in the modulation of early and late gene expression at the transcriptional level (McCarthy et al., 1989). Further analysis demonstrated that Vmw63 performs its regulatory activities over time and mediates these activities indirectly via interactions with, and modification of Vmw175 and possibly other viral and cellular proteins (Rice et al., 1989; '~Present address: Department of Microbiology, The University, Glasgow G12 8QQ, U.K. & Schaffer, 1990). Recent evidence indicates that Vmw63 may be involved at post-transcriptional levels to affect viral mRNA Y processing (Sandri-Goldin & Mendoza, 1992;McLauchlan et al., 1992).In 1987 we reported the isolation of an HSV-1 strain 17 + variant (1703) with a 7'5 kb deletion encompassing part of the long internal repeat region (IRL) and the adjacent unique sequences, such that most of one copy of the IE1 gene, part of UL55 and all of UL56 were deleted (MacLean & Brown, 1987a). The deletion terminate...
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