Human embryo: a biological definition.

This paper defines a human embryo from a biological standpoint that takes into account emerging technologies in reproductive science. The paper does not consider legal, moral, religious or social views. As the definition of a human embryo must reflect the multifactorial processes of development, an approach has been adopted which combines recognition of observed events with potential for further development. This acknowledges that fertilization and development are not static processes, and as such embryo status can only be defined by observation of specific markers. The following biological definition of 'human embryo' is proposed. A human embryo is a discrete entity that has arisen from either: the first mitotic division when fertilization of a human oocyte by a human sperm is complete or any other process that initiates organized development of a biological entity with a human nuclear genome or altered human nuclear genome that has the potential to develop up to, or beyond, the stage at which the primitive streak appears, and has not yet reached 8 weeks of development since the first mitotic division.

PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts.

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 amplified 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 different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification 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 approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.

Recombination signature of germline immunoglobulin variable genes.

In human and mouse, the germline contains a tandem array of highly homologous variable (V) gene elements which encode part of the antigen-binding region of the antibody protein. During evolution this array apparently arose by gene duplication followed by diversification of duplicated genes via point mutation and recombination. Analysis of germline V gene sequences using a novel algorithm shows that major recombination sites coincide with the borders of the leader intron and the cap site, consistent with the hypothesis that over evolutionary time cDNA derived by reverse transcription of pre-mRNA in B lymphocytes has recombined with germline DNA.

The signature of somatic hypermutation appears to be written into the germline IgV segment repertoire.

We present here a unifying hypothesis for the molecular mechanism of somatic hypermutation and somatic gene conversion in IgV genes involving reverse transcription using RNA templates from the V-gene loci to produce cDNA which undergoes homologous recombination with chromosomal V(D)J DNA. Experimental evidence produced over the last 20 years is essentially consistent with this hypothesis. We also review evidence suggesting that somatically generated IgV sequences from B lymphocytes have been fed back to germline DNA over evolutionary time.

Mechanism of antigen-driven somatic hypermutation of rearranged immunoglobulin V(D)J genes in the mouse.

Available data relevant to the mechanism of somatic hypermutation have been critically evaluated in the context of alternative models: (i) error-generating reverse transcription (RT) followed by homologous recombination; and (ii) error-prone DNA replication/repair. A set of basic principles concerning somatic hypermutation has also been formulated and a revised and expanded "RT-Mutatorsome" concept (analogous to telomerase) is presented which is consistent with these principles and all data on the distribution of somatic mutations in normal and Ig transgenic mice carrying particular V(D)J and flanking region constructs. It is predicted that in the mouse VH and Vk loci. the J-C intronic Enhancer-Nuclear Matrix Attachment Region (Ei/MAR) contains a unique sequence motif or secondary structure which ensures that only V(D)J sequences mutate whilst other regions of the genome are not mutated.

Hypothesis: a memory lymphocyte-specific soma-to-germline genetic feedback loop.

Analysis of recently published DNA sequence data obtained for related germline Ig variable (IgV) genetic elements of several vertebrate species revealed the presence of a number of extremely non-random patterns of sequence variability among these genes. Strikingly, the patterns were also observed in two sets of chicken IgV pseudogenes. Since the observed patterns are clearly incompatible with existing theories of multigene family evolution, a new model that can account for all of the data is presented in this paper. The model is a modification and extension of an earlier proposed mechanism whereby somatically expressed genes can be returned to the germline by endogenous retroviruses that may act as soma-to-germline genetic vectors. The mechanism described proposes that the interactions that may result in the soma-to-germline transfer of somatically selected IgV genes occur in the epididymis of the male reproductive tract and are restricted to memory lymphocytes. This mechanism makes a number of predictions that are amenable to experimental testing. From the data presently available in the literature it is not possible to extend the mechanism to the female reproductive tract.

Evolution of V genes: DNA sequence structure of functional germline genes and pseudogenes.

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)

Polymorphic peptide transporters in MHC class I monomorphic Syrian hamster.

We have already shown that in species with highly polymorphic major histocompatibility complex (MHC) class I molecules (human, mouse) no functional polymorphism of the peptide transporters TAP1 and TAP2 is detectable (Lobigs and Müllbacher 1993). Investigating the antigen-presentation machinery of the class I MHC monomorphic Syrian hamster using mouse MHC class I expression via recombinant vaccinia viruses (VV) we found that six hamster cell lines fall into two phenotypic classes. four cell lines (HaK, FF, MF-2, and HT-1) showed no defect in expressing four different H2 class I molecules (Kk, Kd, Kb, Dd) and the appropriate VV peptide recognized by mouse VV-immune cytotoxic T (Tc) cells on the cell surface. Two cell lines (BHK-21 and NIL-2) expressed Dd and Kb in association with VV peptides as recognized by VV-immune, H2-restricted Tc cells but not Kk and Kd. However, Kd was expressed on the cell surface, as shown by fluorescence-activated cell sorter (FACS) analysis and alloreactive Tc-cell recognition. Kk is only surface-expressed in these two cell lines when superinfected with two VV recombinants encoding rat TAP1 (VV-mtp1) and TAP2 (VV-mtp2). Superinfection with VV-mtp1 and VV-mtp2 rendered both cell lines, after infection with either VV-Kk and VV-Kd, susceptible to lysis by either Kk- or Kd-restricted VV-immune Tc cells. Thus Syrian hamster cell lines express functionally polymorphic peptide transporters. The TAP2 gene from FF cells was cloned and sequenced; comparison with human, mouse, and rat TAP2 sequences show 78%, 88% and 87% similarity, respectively.

Analysis of patterns of DNA sequence variation in flanking and coding regions of murine germ-line immunoglobulin heavy-chain variable genes: evolutionary implications.

We analyzed the DNA sequence structure of the 5' flanking and coding regions of 52 VH186.2-related germ-line genes isolated by PCR from C57BL/6J and BALB/c mice. The aligned coding regions display hypervariable and conserved regions corresponding to some of the complementarity-determining regions (CDRs) and framework regions (FRs) found in somatically mutated rearranged immunoglobulin variable genes. Most of the coding regions (88.5%) display open reading frames, strongly suggesting positive selection by antigen. Phylogenetic comparisons of putative transcribed regions versus 5' nontranscribed regions show that they have evolved very differently. Inspection of the 52 murine VH186.2-related DNA sequences (as well as other vertebrate germ-line V sequences reported in the literature) reveals clusters of insertion and deletion events bracketing the transcription/coding unit. These data strongly suggest hyperrecombination events targeting the putative transcription/coding sequence. Given that the DNA of unrearranged germ-line V elements cannot be the direct target for "natural-selection" antigen-binding forces (since V elements are only expressed somatically when rearranged in a mature lymphocyte), it is difficult to explain how these nonrandom sequence variations appear in the germ-line DNA. A number of molecular genetic processes are considered, including antigen-driven soma-to-germ-line gene feedback operative during vertebrate germ-line V gene evolution.

Affinity maturation of lymphocyte receptors and positive selection of T cells in the thymus.

In this review we have re-evaluated the dominant paradigm that TcR V genes do not somatically mutate. We highlight the many structural and functional similarities between Ig and TcR antigen-specific receptors on B and T cells. We have reviewed the factors influencing the somatic and germline evolution of IgV regions in B cells, have evaluated in detail various models which could be invoked to explain the pattern of variation in both transcribed and non-transcribed segments of germline IgV-gene DNA sequences, and applied this perspective to the TcR V beta and V alpha genes. Whilst specific TcRs recognize a complex of a short antigenic peptide bound to MHC Class I or II glycoprotein, and Ig receptors can recognize both oligopeptides and conformational determinants on undegraded polypeptides, they both employ heterodimer variable regions (Fabs) utilizing all three CDRs in epitope binding. We conclude that a plausible case can be made for the possibility that rearranged TcR V genes may undergo some type of somatic hypermutation process during T-cell development in the thymus (concurrent with or after the positive selection phase) thus allowing a repertoire of TvR alpha beta heterodimers to be both positively and negatively selected by the same set of ligands (self MHC + self peptide) in the thymus.

Somatic hypermutation in 5' flanking regions of heavy chain antibody variable regions.

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.

Origin and maintenance of germ-line V genes.

The distribution of nucleotide variability within the upstream of germ-line VH186.2-related variable genes was studied. The data in this report of work in progress indicate non-random selection for variability in the second antigen-contact or complementarity-determining region (CDR2) for 12 such genes isolated using the polymerase chain reaction (PCR) technique from genomic C57BL/6 mouse liver DNA. The translated protein sequences of these and three additional previously published genes also display a pronounced Wu-Kabat peak of amino acid variability in CDR2. In the CDR1 and CDR2 regions of this set of related germ-line genes, there are few [corrected] silent nucleotide changes, and most amino acid replacements (or non-synonymous changes) are non-conservative. In contrast, there is selection against amino acid replacement in the framework regions (FW), as indicated by the significant number of silent (or synonymous) mutational changes from the VH186.2 reference sequence. This is surprisingly similar to the Wu-Kabat variability patterns observed in somatically mutated immune response antibodies. These data could imply similar diversification mechanisms acting on B cell-expressed V genes in the soma (i.e. in a germinal centre) and in the germ-line DNA of male and/or female germ cells. While always possible, we consider this unlikely. Similarly, we consider as unlikely an explanation based on a classical Darwinian model involving simple stepwise whole animal selection prior to reproduction for each VH and VL gene now phylogenetically stored in the V segment arrays of the genomic DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Defining the nucleic acid substrate for somatic hypermutation.

Recent reports have more precisely defined the distribution of somatic mutations around rearranged mouse V-D-J genes. The 5' boundary of mutation is most likely in the region of the transcription start site (cap) and/or the promoter (P), implying that transcription may be a prerequisite for mutations to be generated. As more than 95% of somatic mutations lie downstream of the cap site, the transcription unit itself is implicated as the target of the mutational machinery. For heavy chain genes, the 3' boundary can extend into the enhancer region (E). For kappa light chain genes, the 3' boundary extends to approximately 700 bp beyond J kappa-5 (approximately 700 bp upstream of E). In a single study on mutated derivatives of the rearranged mouse lambda 1 light chain V-J gene, it was claimed that the 3' boundary fell within the constant region (C) exon. Although more data are required, the frequency of mutations around V-D-J genes appears asymmetrical, being positively skewed with a single mode centred near the V-D-J coding region and a long tail extending into the 3' non-translated region of the J-C intron. Such a distribution may place constraints on possible molecular mechanisms. It is suggested that the apparent asymmetrical pattern of mutation is best explained by models that assume localized error-prone DNA synthesis generating variable fragment lengths of mutated DNA or cDNA retrotranscripts. The frequency distribution of these lengths of mutated DNA is positively skewed into the 3' J-C intron, with a common terminus at or near the cap site. It is then assumed that they can be integrated into the target V-D-J region via a gene conversion or homologous recombination process. The model invoking reverse transcription may be preferred as it best explains the data without too many ad hoc assumptions.