Summary Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be ampli®ed simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between dierent germline sequences during ampli®cation are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of ampli®cation cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of ampli®cation cycles, becoming a high proportion of the total number of PCR products once the`plateau phase' of the reaction was reached. Recombination events were located throughout the $ 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of ampli®cation cycles), recombination artefacts can be virtually eliminated from PCR ampli®cations involving mixtures of very similar sequences. This information is relevant to all studies involving PCR ampli®cation of members of highly homologous multigene families of cellular or viral origin.

The aim of this study has been to determine the distribution of somatic mutations in the 5' flanking regions of rearranged immunoglobulin heavy chain variable region genes (VDJ). We sequenced the 5' flanking region in 12 secondary immune response antibodies produced in C57BL/6j mice against the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken-gamma-globulin. In these and previously published sequences, almost 97% of the mutations occurred in the transcribed region of the gene, and only a minority of genes (5/29) contained mutations upstream of the transcription start (cap) site. No potential germ-line donor was found for a cluster of five base changes previously found in a single heavy chain gene, 3B62. However, the uniqueness of this mutational cluster and its distance from the normally mutated region suggests that the nucleotide changes may not be due to the normal mutator mechanism. Thus, as this was the only instance of somatic mutations that far upstream of the promoter/cap site region, the reverse transcriptase model for somatic hypermutation is still a possibility. The data are consistent with a mutational mechanism that requires transcription of the rearranged target V(D)J gene which appears to result in the generation of a positively skewed asymmetrical distribution of somatic mutations. A single mode is centered near the V(D)J and a long tail extends into the 3' non-translated region of the J-C intron. Two classes of model could explain this mutation distribution pattern: those where transcription products (RNA, cDNA) are the direct mutational substrates, or those that postulate local unfolding of the chromatin around a V(D)J rearrangement directly exposing the DNA of the transcribed region to specific mutational enzymes.

In this review we have examined the features of germline sequences of IgV genes from a number of species in an attempt to identify the "signature" of molecular mechanisms responsible for generating and maintaining diversity in the germline repertoire (after gene duplication by meiotic unequal crossover). We now summarize the relevant features point by point: 1. Codon analysis reveals a significant deficit of stop codons below the numbers that would be expected under random point mutational change. This implies that the majority of individual V genes have each been selected for the possession of open reading frames able to encode a functional Ig molecule. There is an extraordinarily high rate of apparent rescue of potential stop codons in both V genes and pseudogenes. Other (non-Ig) pseudogene sequences studied thus far do not show this high rate of rescue of stop codons. 2. The distribution of changes is concentrated in most cases in the 5' half of CDR2 (CDR2a), and coincides with the patterns of antigen-selected mutations in B lymphocytes. It does not coincide with expected non-antigen-selected (random) changes, as exemplified by hypermutated but unexpressed passenger V transgenes in B cells in Peyer's patches of unimmunized mice (Gonzalez-Fernandez and Milstein 1993). 3. In germline V genes of mice, there is no evidence of triplet codon insertion (or multiples thereof) as a mechanism generating germline diversity. This parallels a known absence of gene conversion as a mechanism generating somatic diversity in mice. In contrast, in germline chicken pseudogenes which are known to contribute to somatic generation of diversity by gene conversion, frequent examples of triplet codon insertions and deletions in CDRs are present. 4. The pattern of unique insertions and deletions in all species with sufficient sequence data available is consistent with hyper-recombination events targeting the transcription and/or coding unit. The distribution of these events does not correlate with known inducers of gene conversion, for example, inverted or direct repeats and palindromes. Furthermore, the 5' boundaries of somatic hypermutation and the 5' peak of germline nucleotide insertions and deletions coincide in IghV (Rothenfluh et al. 1993, 1994; Rogerson 1994) and in IgkV (Rogerson 1994; Rada et al. 1994, and analyses herein). It will be interesting to see how these features relate to each other in other gene sets as data become available.(ABSTRACT TRUNCATED AT 400 WORDS)

Alphaviruses, such as chikungunya virus, o'nyong-nyong virus, and Ross River virus (RRV), cause outbreaks of human rheumatic disease worldwide. RRV is a positive-sense single-stranded RNA virus endemic to Australia and Papua New Guinea. In this study, we sought to establish an in vitro model of RRV evolution in response to cellular antiviral defense mechanisms. RRV was able to establish persistent infection in activated macrophages, and a small-plaque variant (RRV PERS ) was isolated after several weeks of culture. Nucleotide sequence analysis of RRV PERS found several nucleotide differences in the nonstructural protein (nsP) region of the RRV PERS genome. A point mutation was also detected in the E2 gene. Compared to the parent virus (RRV-T48), RRV PERS showed significantly enhanced resistance to beta interferon (IFN-␤)-stimulated antiviral activity. RRV PERS infection of RAW 264.7 macrophages induced lower levels of IFN-␤ expression and production than infection with RRV-T48. RRV PERS was also able to inhibit type I IFN signaling. Mice infected with RRV PERS exhibited significantly enhanced disease severity and mortality compared to mice infected with RRV-T48. These results provide strong evidence that the cellular antiviral response can direct selective pressure for viral sequence evolution that impacts on virus fitness and sensitivity to alpha/beta IFN (IFN-␣/␤).Arthritogenic alphaviruses are distributed globally and are maintained in nature by cycles of transmission between hematophagous arthropods (for example, mosquitoes) and enzootic vertebrate hosts (mammals, marsupials, or birds) (33, 53). Nearly all symptomatic infections with these alphaviruses manifest with joint symptoms (arthritis and arthralgia), with myalgia, rash, and lethargy also being common. Ross River virus (RRV) is an Australian alphavirus associated with chronic polyarthritis and causes up to 8,000 cases annually in Australia, with the number of cases reported in new localities increasing (42,55). During the late 1970s and early 1980s, a number of South Pacific island nations experienced a major outbreak of RRV disease (RRVD) affecting more than 50,000 people (17). More recently, a related virus, chikungunya virus (CHIKV), caused similar rheumatic disease in one-third of the population of Réunion Island in the Western Indian Ocean and an estimated 1.39 million cases in India (22,23,37,52). This outbreak was also associated for the first time with some severe clinical manifestation and mortality (37).In RRV-infected patients, the disease is usually severe at onset with a gradual resolution over 3 to 6 months (40). Alphavirus-induced rheumatic disease is thought to have a substantial immunopathological component. For example, in animal models tissue damage results from the induction of proinflammatory cytokines and chemokines and recruitment of macrophages in response to infection (29,43,55). RRV infects and replicates in human and mouse macrophages, and infection of the mouse macrophage cell line RAW 264.7 results in the establishment of ...