Germline genomic structure of the B10.A mouseTcra-V2 gene subfamily

 The number of mouse Tcra-V gene segments varies from one individual to another and is estimated to be about 100. Southern blot analysis revealed that most of the Tcra-V are organized in clusters composed of copies of Tcra-V belonging to different subfamilies. We analyzed in detail a Tcra-V subfamily and looked for new Tcra-V in order to improve the knowledge of the mouse Tcra locus organization. A series of genomic clones derived from the B10.A mouse strain enclosing these clusters was used to determined the structure of all the Tcra-V2. We were able to identify ten Tcra-V2. This study showed that the Tcra-V2 can be organized into three structural subgroups. The distribution of the genes along the Tcra locus, plus their structural organization, indicates that successive duplications occurred during the processes of expansion and contraction of the Tcra-V gene subfamilies. Several Tcra-V2 are also identical, indicating recent duplications. The most divergent Tcra-V2 differ by 7.4% nucleotides, leading to 5.2% differences in amino acid contents. Received: 8 August 1995 / Revised: 24 April 1996     

Chromosome 14 in B10.A(18R) mice is recombinant and includes Tcra-V a alleles

Analysis of mouse Tcr genes has previously defined at least five different Tcra-V haplotypes among inbred strains of mice. For mice of the Tcra-V ^b haplotype, including C57BL/10 (B10), T-cell expression of the Tcra-V11 gene subfamily can be detected with a monoclonal antibody, 1.F2. In the course of further characterizing the specificity of 1.F2, we found that it fails to recognize Tcra-V11-expressing T-cell hybrids derived from the B10 congenic strain, B10.A(18R)/SgIcr. Moreover, staining analysis indicated that the Va11 epitope recognized by 1.F2 is not expressed by peripheral T cells from several different B10.A(18R) colonies with the exception of that at the Research Institute of Scripps Clinic. Nucleotide sequences were determined for cDNA representing rearranged Tcra-V11 genes from two independent, B10.A(18R)/SgIcr derived T-cell hybrids. The two Tcra-V11 gene segments were identical and the predicted amino acid sequence differed by at least five residues from Tcra-V11 sequences previously obtained from B10.A mice. Southern blot analysis of restriction fragment length polymorphisms (RFLP) associated with Tcra-V11, as well as Tcra-V ₁ , subfamily genes revealed that the B10.A(18R) mouse has inherited Tcra-V ^a alleles rather than the expected Tcra-V ^b alleles from the B10 strain. RFLP analysis of the Rib-1 locus, located in close proximity to the Tcra locus on chromosome 14, showed that B10.A(18R) carries the Rib-1 ^b allele from B10. These results indicate that the B10.A(18R) mouse has inherited a recombinant chromosome 14 with a recombination event having occured between the Rib-1 locus and the Tcra-V ^a gene subfamilies examined. Inheritance of Tcra-V ^a alleles in B10.A(18R) probably originated from strain 129/J which breeding records show was used in the first cross with B10.A in the production of B10.A(18R) and which we found exhibits Tcra-V11 ^a RFLPs.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M55634 and M55635. Address correspondence and offprint requests to: P. B. Nakajima.     

Diversity of the Tcra-V3 gene family in BALB/c mice

 Southern analysis of Eco RI-digested BALB/c liver DNA reveals four T-cell receptor Tcra-V3-hybridizing DNA fragments, which are of sizes 18.0, 12.0, 8.0, and 2.1 kilobases, respectively. These four Tcra-V3-hybridizing genomic DNA were isolated from a BALB/c genomic library. Restriction and Southern analysis of the genomic DNA clones showed that each of the Tcra-V3-hybridizing Eco RI DNA fragments harbors only a single Tcra-V3 gene. The DNA sequences of coding regions of the four Tcra-V3 family members were determined. These sequences show very limited divergence from one another. Comparisons of BALB/c Tcra-V3 sequences with published Tcra-V3 sequences expressed in different strains of mice reveal substantial allelic polymorphism. Sequence similarity searches retrieved homologous rat, cattle, and human genes. The scarcity of coding sequence divergence among members of the Tcra-V3 family and the more substantial allelic polymorphism may be general features of the T-cell receptor V-alpha chain-encoding gene families. Received: 11 April 1996 / Revised: 26 May 1996     

No recombinations between Tcra-V and Tcra-C gene segments in 669 backcross mice

Because T-cell receptor (Tcr) genes may possibly function as non-major histocompatibility complex (MHC) immune response genes or predispose for autoimmune diseases, it is important to know how these genes are inherited. We found that Bgl I-digested DNA of BALB/c, C3H, DBA/2, and C57BL/6 exhibited restriction enzyme fragment length polymorphisms (RFLPs) for the Tcra-V1, Tcra-V2, Tcra-V4, Tcra-V6, Tcra-V7, Tcra-V8, Tcra-V11, Tcra-122, Tcra-V13, and Tcra-C gene segments. Inheritance of these RFLPs in 669 offspring from (BALB/c × C57BL/6) × BALB/c, (BALB/c × C57BL/6) × C57BL/6, (C57BL/6 × DBA2) × DBA/2, and (C57BL/6 × C3H) × C3H backcrosses was studied. Since we did not find any recombinations in the offspring, Tcra-V and Tcra-C gene segments are tightly linked and inherited as a haplotype. A peculiar finding was that 22 out of 103 (BALB/c × C57BL/6) × BALB/c offspring, heterozygous for Tcra-C, had deleted a C57BL/6 Tcra-V1 band as well as Tcra-V2 and Tcra-V4 bands. As will be discussed, this deletion is probably caused by heterogeneity in the C57BL/6 breeding stock of a commercial supplier. In seven BXD and BXH recombinant inbred strains with known recombinations between the Tcra-C and Es-10 loci, all Tcra-V RFLPs cosegregated with the Tcra-C RFLP. This finding agrees with the conclusion from our backcross studies; namely that Tcra-V and Tcra-C gene segments are tightly linked.     

Comparison of human and mouse T-cell receptor variable gene segment subfamilies

Like the immunoglobulin Igh-V and Igk-V gene families, the human or mouse TCRV gene families may be grouped into subfamilies displaying >75% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most mouse Tcr-V subfamilies exhibit clear homology to human TCRV subfamilies (>60% amino acid sequence similarity). Homologous pairs of TCRV genes in mice and humans show higher sequence similarity than TCRV genes from different subfamilies within either species, indicating transpecies evolution of TCRV genes. Mouse and human homologues show conservation of their relative map order, particularly in the 3' region and a similar sequential and developmentally programmed expression. When the V regions from both species were analyzed together, local length differences and conserved residues in the loop regions were revealed, characteristic of each of the four TCRV families.The alignment data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the alignment number DS23485. The data are available by the EBI FTP server and file server     

FVB/N (H2 q ) mouse is resistant to arthritis induction and exhibits a genomic deletion of T-cell receptor V beta gene segments

 Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis (CIA) in mice is an autoimmune disease model of rheumatoid arthritis. Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). CD4⁺ T cells that express the T-cell receptor (TCR) Tcra-V11.1 and/or Tcrb-V8.2 play a key role in the pathogenesis of arthritis in the DBA/1 mouse (H2 ^q ). We identified an inbred mouse strain, FVB/NJ (H2 ^q ), that is resistant to arthritis induction and exhibits a genomic deletion of certain Tcrb-V gene segments. We report a novel polymerase chain reaction-based method for the rapid identification of new mouse strains that exhibit germline Tcrb-V gene deletions. We mapped for the first time both the 5′ and 3′ breakpoints of the Tcrb-V deletion in the FVB/NJ, SWR, SJL, C57L, and C57BR strains to within 1.1 kilobases. Since there is an association between a particular Tcra-V allele (Tcra-V11.1 ^d ) and arthritis susceptibility in H2 ^q mouse strains, we examined the allelic polymorphisms of the Tcra-V11 gene subfamily members between the arthritis-susceptible DBA/1 mouse and the arthritis-resistant FVB/NJ mouse strain. The amino acid sequences of the Tcra-V11.1 alleles differ at two positions (codons 18 and 68). Therefore, the resistance of FVB/NJ mouse to arthritis induction may be due in part to Tcra-V11.1 coding sequence polymorphism and Tcrb-V8.2 gene segment deletion, as we have recently demonstrated in the case of SWR mouse strain. Received: 12 January 1999 / Revised: 17 March 1999     

Organization, polymorphism, and expression of the human T-cell receptor AV1 subfamily

 At least 32 mostly single-member subfamilies of T-cell receptor alpha variable (TCRAV) genes have been described in humans. The AV1 subfamily is the largest, estimated by hybridization to contain as many as five members. However, a search of nucleotide sequence databases reveals a much greater number of unique sequences corresponding to this subfamily. In order to resolve this discrepancy between hybridization and nucleotide sequencing data, and to better understand the nature of variability among variable genes within a large subfamily, a genomic characterization of the AV1 subfamily in humans was carried out. Total genomic DNA, as well as isolated genomic clones spanning the TCRA region were screened for members of the AV1 subfamily by polymerase chain reaction (PCR) and nucleotide sequencing as well as by hybridization. A total of eight AV1 genes were identified and their nucleotide sequences were determined. Three of the sequences represent new genes. Based on structural features and the results of PCR screening of cDNA, none of these new genes appear to be functional. Several additional previously reported AV1 sequences were determined to represent alleles of AV1 genes, and simple PCR restriction digest assays were established for their detection. Use of each of the identified AV1 genes as hybridization probes failed to reveal any additional hybridizing bands. Thus the AV1genes represent the largest TCRAV subfamily with a maximum of eight members, several of which have common allelic forms. Received: 7 November 1996 / Revised: 5 December 1996     

Structural analysis of the mouse T-cell receptor Tcra V2 subfamily

Cosmid clones containing T-cell receptor Tcra V2 subfamily gene segments have been isolated from a BALB/c cosmid library and subjected to DNA sequence analysis. The V gene segments in the Tcra V2 subfamily differ from each other by 3%–7% at the nucleotide level and 5%–16% at the amino acid level. T-cell receptor Tcra V2 gene segment polymorphisms have been identified in the B10.PL and PL/J mouse strains with a Tcra V2 subfamily-specific probe. These V gene segment polymorphisms may cause the differential Tcra V gene usage in induced experimental allergic encephalomyelitis between B10.PL and PL/J mice.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession numbers U04312 and U04622-U04626     

Cloning, localization, and structure of new members of the butyrophilin gene family in the juxta-telomeric region of the major histocompatibility complex

 New members of the butyrophilin (BT) gene family have been identified by cDNA and genomic cloning. Six genes are described: BT2.1, 2.2, 2.3, and BT3.1, 3.2, and 3.3. BT2, BT3, and BT form three distinct subfamilies sharing about 95% amino acid identity at the intra subfamily level and 50% identity at the interfamily level. All the BT2 and BT3 subfamily members map close to BT in the juxta-telomeric region of the major histocompatibility complex. The BT2 members have the canonical structural organization of BT, i.e., two immunoglobulin domains followed by a transmembrane anchor and a B30.2 intracytoplasmic domain. In the BT3 subfamily, only BT3.3 has the structural organization of BT. The two other genes, BT3.1 and BT3.2, code for putative protein without the B30.2 domain. In the case of BT3.2, this is due to an Alu insertion in the B30.2 coding exon, leading to a new polyadenylation site. Received: 21 April 1997 / Revised: 13 June 1997     

Organization of the major latex protein gene family in opium poppy

Opium poppy latex contains a group of laticifer-specific, low-molecular-weight polypeptides called major latex proteins (MLPs). Here we describe a new member of the MLP gene family (gMLP 22) which shares 79.6% nucleotide and 84.6% amino acid sequence identity with previously isolated clones. DNA gel blot analysis indicates that the MLPs are encoded by at least eight genes which are divided into two subfamilies. The expression pattern for each subfamily, as seen in RNA gel blots, appears to be identical and laticifer-specific.     

Molecular analysis of skewed Tcra-V gene use in T-cell receptor β-chain transgenic mice

 The influence of β-chain diversity on the expressed T-cell receptor (TCR) α-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR β-chain gene. Analysis of these mice using α-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded β chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards α chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in β-transgenic mice is exhibited almost exclusively by CD4⁺ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8⁺ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed β chains can profoundly influence the selection of companion α chains expressed in the periphery, and that α-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3α domain participates in direct contact with the MHC. Received: 28 January 1997 / Revised: 22 July 1997     

PCR-based genotyping and haplotype analysis of human TCRBV gene segment polymorphisms

There are at least 63 tandemly arranged human T-cell receptor (Tcr) -chain variable region (BV) gene segments, which have presumably arisen by repeated gene duplication events. The 5-most half of the TCRBV gene loci is particularly complex in organization due to the presence of multiple interspersed members of the largest BV subfamilies, BV5, BV6, and BV13. Polymorphism and linkage relationships among these genes has been poorly characterized in part due to the high similarity of these duplicands. Germline DNA polymorphisms were specifically examined in the exons and introns of these and other BV gene segments distributed across 240 kilobases (kb) in this 5-most region. Polymerase chain reaction restriction enzyme-based assays were used to genotype ten point mutations in seven of the BV gene segments. Eight of these polymorphisms altered an amino acid of the BV gene segment. In addition, length polymorphisms due to simple sequence repeats were noted in the introns of six BV6 subfamily members. Approximately 250 unrelated haplotypes were constructed by segregation analyses of fifteen of these TCRBV polymorphisms. Linkage disequilibrium analyses indicated that haplotypic relationships are not detectable over a distance of more than 55 kb in this genomic region. These TCRBV polymorphisms, and the haplotypic analysis, provide important resources and guidance for future attempts to associate Tcr germline DNA differences in the human population with immune response differences, such as might occur in some autoimmune diseases.     

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5 and 3 flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.     

T-cell receptor genes in tassel-eared squirrels (Sciurus aberti)

The role of environmental factors in the evolution and maintenance of diversity of antigen receptor gene families which participate in the immune response in mammals is inadequately understood. In order to elucidate the impact of these factors, we have undertaken the analysis of these gene families in the tassel-eared squirrel (Sciurus aberti) which has been separated into discrete subspecies by geographic barriers and whose food resources can be quantitated for estimating environmental quality. In this communication we describe the initial analysis of the complexity and polymorphism of sequences related to T-cell receptor (Tcr) and chain genes in two subspecies, Sciurus aberti aberti (Abert) and Sciurus aberti kaibabensis (Kaibab) which have identical habitats and are separated by the Grand Canyon in Arizona, USA. Genomic blot analysis of 60 Abert and 62 Kaibab individuals collected over a 3-year period was performed with mouse Tcrb and Tcra cDNA probes. Sequences homologous to Tcrb-C, Tcrb-J1, and Tcrb-J2 genes were observed in all individuals from both subspecies; although Tcrb-J1 fragments were monomorphic, Tcrb-C and Tcrb-J2 fragments were polymorphic with both species- and subspecies-specific sequences. A single, monomorphic Tcra-C fragment was observed in addition to multiple Tcra-V fragments homologous to the mouse Tcra-V1 subfamily. Abert samples exhibited greater numbers of Tcra-V1 fragments as well as greater polymorphism than Kaibab samples. Heterozygosity estimates of Tcrb-C and Tcra-V1 sequences were determined for annually collected samples and compared with the yearly estimates of availability of hypogeous fungi, one of the major diet items of tassel-eared squirrels. In the Kaibab annual collections, Tcra-V1 heterozygosity declined with the decline in food resource, whereas heterozygosity of Tcrb-C sequences was inversely related to food resources. Similarly, a reduction in food resource for Abert squirrels in 1985 coincided with an increase in Tcrb-C heterozygosity in the same year. These results suggest that the diversity of gene families which participate in the immune response in mammals may be affected by environmental factors. Address correspondence and offprint requests to: P. J. Wettstein.     

Diversity, Distribution, and Ancient Taxonomic Relationships Within the TIR and Non-TIR NBS-LRR Resistance Gene Subfamilies

Phylogenetic relationships among the NBS-LRR (nucleotide binding site–leucine-rich repeat) resistance gene homologues (RGHs) from 30 genera and nine families were evaluated relative to phylogenies for these taxa. More than 800 NBS-LRR RGHs were analyzed, primarily from Fabaceae, Brassicaceae, Poaceae, and Solanaceae species, but also from representatives of other angiosperm and gymnosperm families. Parsimony, maximum likelihood, and distance methods were used to classify these RGHs relative to previously observed gene subfamilies as well as within more closely related sequence clades. Grouping sequences using a distance cutoff of 250 PAM units (point accepted mutations per 100 residues) identified at least five ancient sequence clades with representatives from several plant families: the previously observed TIR gene subfamily and a minimum of four deep splits within the non-TIR gene subfamily. The deep splits in the non-TIR subfamily are also reflected in comparisons of amino acid substitution rates in various species and in ratios of nonsynonymous-to-synonymous nucleotide substitution rates (K _A/K _S values) in Arabidopsis thaliana. Lower K _A/K _S values in the TIR than the non-TIR sequences suggest greater functional constraints in the TIR subfamily. At least three of the five identified ancient clades appear to predate the angiosperm–gymnosperm radiation. Monocot sequences are absent from the TIR subfamily, as observed in previous studies. In both subfamilies, clades with sequences separated by approximately 150 PAM units are family but not genus specific, providing a rough measure of minimum dates for the first diversification event within these clades. Within any one clade, particular taxa may be dramatically over- or underrepresented, suggesting preferential expansions or losses of certain RGH types within particular taxa and suggesting that no one species will provide models for all major sequence types in other taxa. Received: 13 June 2001 / Accepted: 22 October 2001     

Evolution of the βGRP/GNBP/β-1,3-glucanase family of insects

The βGRP/GNBP/β-1,3-glucanase protein family of insects includes several proteins involved in innate immune recognition, such as the β-glucan recognition proteins of Lepidoptera and the Gram-negative bacteria-binding proteins of Drosophila. A phylogenetic analysis supported the existence of two distinct subfamilies, designated the pattern recognition receptor (PRR) and glucanase subfamilies, which originated by gene duplication prior to the origin of the Holometabola. In the C-terminal region (CTR) shared by both subfamilies, the PRR subfamily has evolved significantly more rapidly at the amino acid sequence level than has the glucanase subfamily, implying a relative lack of constraint on the amino acid sequence of this region in the PRR subfamily. PRR subfamily members also include an N-terminal region (NTR), involved in carbohydrate recognition, which is not shared by glucanase subfamily members. In comparisons between paralogous PRR subfamily members, there were no conserved amino acid residues in the NTR. However, when pairs of putatively orthologous PRR subfamily members were compared, the NTR was most often as conserved as the CTR or more so. This pattern suggests that the NTR may be important in functions specific to the different paralogs, while amino acid sequence changes in the NTR may have been important in functional differentiation among paralogs, specifically with regard to the types of carbohydrates that they recognize.     

Proteins of theXenopus laevis zinc finger multigene family as targets for CK II phosphorylation

Zn finger proteins (ZFPs) of the C₂/H₂ type inXenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XIcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites forin vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activityin vivo is discussed.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

A new Igk-V gene family in the mouse

We have identified and characterized a novel mouse Igk-V gene family, which we have designated Igk-V34. Southern hybridization and nucleotide sequence analysis indicate that this family is comprised of either one or two members in mice of different Igk haplotypes. The gene family members share between 95% and 98% sequence similarity, indicating that they diverged only recently during the evolution of the Igk locus. Sequence relationships between members of this family are discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M35154-7. Offprint requests to: A. J. Caton.