The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.

We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.     

Mechanisms underlying telomere repeat turnover, revealed by hypervariable variant repeat distribution patterns in the human Xp/Yp telomere.

Sequences immediately adjacent to the human Xp/Yp telomere exhibit a high frequency of base substitutional polymorphisms, together with almost complete linkage disequilibrium, to create only a few diverged haplotypes. This sequence divergence has been used to develop a PCR-based system for mapping the distribution of the telomere (TTAGGG) and variant repeats (TGAGGG and TCAGGG) at the proximal end of the telomere repeat array. The distribution of these repeats is extremely variable. Almost all Xp/Yp telomeres are different, indicating a high mutation rate. Some telomere maps associated with the same flanking haplotype show similarities, identifying subsets of telomeres that share a recent common ancestry. Mechanisms underlying the rapid turnover of repeats at the proximal end of the Xp/Yp telomere include intra-allelic processes, such as slippage during replication. Inter-allelic exchanges may occur occasionally, but telomerase activity probably plays only a minor role in the germline turnover of proximally located telomere and variant repeats.     

Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens.

In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.     

Origins of polymorphism at a polypurine hypervariable locus.

We present characterisation of a hypervariable locus, D8S210, mapped to the telomeric region of the short arm of chromosome 8. The locus is highly polymorphic with alleles varying in size from 1.8 kb to 24 kb. Sequence data from 7 alleles shows that the variable region is entirely polypurine on one strand with a tetranucleotide repeating unit GGAA at the margins and diverged versions of this motif internally. The margins are conserved between alleles; polymorphism occurring in the internal regions of the repeat. Alleles are inherited in a Mendelian manner and one new mutation has been observed in analysis of 51 meioses. Use of single copy flanking sequences to elaborate the polymorphism revealed loss of single copy DNA in 3 unrelated families and in 2 other unrelated individuals. Restriction mapping shows that this loss is similar for different sized alleles in all three families suggesting that it was an early event that may have involved a flanking Alu sequence. We present evidence that the polypurine region can adopt triplex conformations in vitro. Such structures may facilitate loss or gain of unique sequences in the genome, contribute to mutation at conformation transition points and drive the hypervariability (> 99% heterozygosity) of this locus.     

A novel GC-rich dispersed repeat sequence in Drosophila melanogaster.

A novel family of dispersed repeat sequences from Drosophila melanogaster is described. Sequence analysis of two members of this family show them to contain greater than 75% GC bases. These are comprised of multiple repeats of GGX triplets interspersed occasionally with CGPy and TTPy. Southern blotting shows that these repeats are not transposable elements. Twenty four homologous recombinants have been localised by in situ hybridization to seven sites in the Drosophila genome. Polyadenylated RNAs homologous to this repeat family are expressed in a complex pattern which is developmentally regulated. We suggest that this family encodes a set of glycine-rich domains in Drosophila proteins.     

Extensive telomere repeat arrays in mouse are hypervariable.

In this study we have analysed mouse telomeres by Pulsed Field Gel Electrophoresis (PFGE). A number of specific restriction fragments hybridising to a (TTA-GGG)4 probe in the size range 50-150kb can be detected. These fragments are devoid of sites for most restriction enzymes suggesting that they comprise simple repeats; we argue that most of these are likely to be (TTAGGG)n. Each discrete fragment corresponds to the telomere of an individual chromosome and segregates as a Mendelian character. However, new size variants are being generated in the germ line at very high rates such that inbred mice are heterozygous at all telomeres analysable. In addition we show that specific small (approximately 4-12kb) fragments can be cleaved within some terminal arrays by the restriction enzyme MnII which recognises 5'(N7)GAGG3'. Like the complete telomere-repeat arrays (TRA's) these fragments form new variants at high rates and possibly by the same process. We speculate on the mechanisms that may be involved.     

Mutation rate in the hypervariable VNTR g3 (D7S22) is affected by allele length and a flanking DNA sequence polymorphism near the repeat array.

The hypervariable human minisatellite locus D7S22 (g3) is highly polymorphic. The allelic distribution in D7S22 features a size clustering of the alleles and a comparably low allelic diversity among small alleles. This reduced diversity could reflect a situation where some alleles are less likely to mutate than others. Several factors could explain such an effect, including allele size, variation in repeat composition, and allelic differences in nearby cis-acting elements affecting the mutation rate. We have characterized 40 de novo mutations found on Southern blots in a large amount of paternity-testing material. There is a significant excess of paternal mutations, and small size changes are most frequent. Mutation rate is affected by allele length, with highest rates in larger alleles. Alleles of the family groups with D7S22 mutations and 50 small alleles were analyzed by nucleotide sequencing. Two hundred thirty-six base pairs of the immediate flanking region upstream of the repeat array were PCR amplified and screened for point mutations by DNA sequencing of the PCR products. Two base substitution polymorphisms were identified: one C/G transversion and one A/G transition, 54 bp and 173 bp upstream of the repeat array, respectively. There is a significant association between mutation and occurrence of 54C, while association is not obvious between mutation rate and the 173A/G variants. There is a marked association between different flanking haplotypes and allele size, and within the smallest allele-size group, all alleles had the 54G/173A haplotype. Both allele size and allelic state at site 54 remain associated with mutation rate when the other factor is controlled. Possible mechanisms behind the variation in mutation rate in D7S22 are discussed.     

Characterization of MboI repeat DNA sequence of Anopheles stephensi.

MboI repeat fragment of mosquito Anopheles stephensi has been isolated by molecular cloning. The restriction map and entire nucleotide sequence of the 433bp insert has been determined. Hybridization of this repeat DNA with restriction enzyme digest of mosquito DNA does not show an interspersed pattern but suggests that this repeat may be tandemly repeated at one major site and a few minor sites in the genome of Anopheles stephensi. The hybridization pattern also indicates that this repeat family comprises of many similar but non-identical sequences. An open reading frame encoding 66 amino acids with an initiation and two tandem termination codons has been identified. This putative 66 amino acid polypeptide sequence has significant homology to a small region of RNA tumour viral envelope protein.     

Complete genome sequence of a novel type of human parechovirus strain reveals natural recombination events

Human parechoviruses (HPeVs) are a species in the Parechovirus genus of the Picornaviridae family. We report a complete genome sequence of a novel HPeV strain, CH-ZJ1, that was found in an infant with gastroenteritis in Zhenjiang City, China. The complete genome consists of 7,298 nucleotides (nt), excluding the 3' poly(A) tail; the open reading frame is mapped between nucleotide positions 654 and 7211 and encodes a 2,185-amino acid (aa) polyprotein. The phylogenetic tree obtained for the complete genome of this HPeV strain and the other HpeV strains available in GenBank indicated that CH-ZJ1 is intervenient between HpeV type 4 (HpeV4) and HpeV5. Phylogenetic analysis based on the 3D and VP1 genes reveals two incongruent trees. Recombination detection indicated that CH-ZJ1 might be a recombinant which was produced by more than one genomic recombination event that occurred among HPeV1, HPeV4, and HPeV3 strains.     

Rat mitochondrial DNA polymorphism: sequence analysis of a hypervariable site for insertions/deletions

Three closely related variants of rat (Rattus norvegicus) mtDNA have been shown to differ in the number of T residues found in a run of Ts (light strand) which spans the junction between the tRNACys and tRNATyr genes. The number of Ts in the repeat varies from 6 to 8 in these DNAs. Another, less closely related, R. norvegicus variant has a run of 5 Ts at this site and in the related species, Rattus rattus, a run of 4 Ts is found. In R. norvegicus mtDNA runs of 5 As and 5 Gs are found just to the 3' side of the variable T repeat, and it is suggested that the three runs of repeated nucleotides may stabilize heteroduplexes which result from strand slippage and which give rise to the insertions and/or deletions. Among 17 mtDNA clones derived from an individual with the 8T repeat, one clone was found which possessed a 9T repeat. This variant may represent an additional DNA type originally present within the individual.     

CAG repeat instability, cryptic sequence variation and pathogeneticity: evidence from different loci.

Different aspects of expanded polyglutamine tracts and of their pathogenetic role are taken into consideration here. (i) The (CAG)n length of wild-type alleles of the Huntington disease gene was analysed in instability-prone tumour tissue from colon cancer patients to test whether the process leading to the elongation of alleles towards the expansion range involves single-unit stepwise mutations or larger jumps. The analysis showed that length changes of a single unit had a relatively low frequency. (ii) The observation of an expanded spinocerebellar ataxia (SCA)1 allele with an unusual pattern of multiple CAT interruptions showed that cryptic sequence variations are critical not only for sequence length stability but also for the expression of the disease phenotype. (iii) Small expansions of the (CAG)n sequence at the CACNA1A gene have been reported as causing SCA6. The analysis of families with SCA6 and episodic ataxia type 2 showed that these phenotypes are, in fact, expressions of the same disorder caused either by point mutations or by small (CAG)n expansions. A gain of function has been hypothesized for all proteins containing an expanded polyglutamine stretch, including the alpha 1A subunit of the voltage-gated calcium channel type P/Q coded by the CACNA1A gene. Because point mutations at the same gene with similar phenotypic consequences are highly unlikely to have this effect, an alternative common pathogenetic mechanism for all these mutations, including small expansions, can be hypothesized.     

Characterization of a single nucleotide polymorphism in the coding sequence of the bovine transferrin gene.

A single nucleotide polymorphism was identified in the coding sequence of the bovine transferrin gene. Two alleles (SSCP1 and SSCP2) were detected by SSCP analysis and the mutation point was identified and confirmed by direct sequencing of the PCR products. The relationship between protein and DNA polymorphism was established. Protein variants A, D1 and E correspond to SSCP allele 1 and variant D2 corresponds to SSCP allele 2. DNA sequences from genotypes AA, AE, AD2, D1E, D2E and D2D2 reveal an A/G substitution at position 1455 of the cDNA which causes a Gly/Glu substitution which could be responsible for the mobility difference between D1 and D2 variants. Because of the number of variants, this suggests that other SNPs exist in the bovine transferrin gene. A linkage analysis between the SSCPs and two microsatellites (UWCA46 and CSSM019) mapped the transferrin gene to BTA1. Two-point analysis revealed a tight linkage within the transferrin protein variants and the SSCPs.     

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.     

Identification of a hypervariable region in the long terminal repeat of equine infectious anemia virus.

An avirulent, field-derived isolate of equine infectious anemia virus (EIAV), designated MA-1, was molecularly cloned, and the complete nucleotide sequence was determined for the 3' half of the viral genome. Comparisons between MA-1 and the prototype Wyoming strain of EIAV identified a 66-nucleotide stretch between CAAT (-91) and TATAA (-25) in the U3 region of the long terminal repeat, where sequence divergence was as high as 39.3%. The polymerase chain reaction was used to amplify and clone long terminal repeat sequences from Th-1, the in vivo parental stock of MA-1. Results indicated that the nucleotide sequences of MA-1 and Th-1 clones were less variable than was observed between MA-1 and Wyoming. However, MA-1 and Th-1 markedly differed in the types of enhancer sequences located in the hypervariable region. These results suggest that variation in lentivirus regulatory sequences may be important in EIAV host cell tropism and pathogenesis.     

Polymorphic microsatellite and cryptic simple repeat sequence markers in pineapples (Ananas comosus var. comosus)

A rapid method for isolating microsatellite loci in pineapples, based on the 5′‐anchored polymerase chain reaction technique, revealed 137 microsatellite loci (consisting of 62 dinucleotide, 24 trinucleotide, 49 tetranucleotide and 2 hexanucleotide repeats) and 16 cryptically simple repeat sequences. We report on the characterization of 19 polymorphic microsatellite loci and one cryptic simple repeat loci in pineapples. The number of alleles per locus ranged from two to four while the observed heterozygosity ranged from 0.1705 to 1. These markers are useful as tools for detecting levels of genetic variation in pineapple varieties for germplasm management and crossbreeding purposes.     

Unsuspected cryptic polymorphism in the polychromatic Midas cichlid.

The Midas cichlid ( Cichlasoma citrinellum ) of Nicaragua shows stable polychromatism in that a consistent, small proportion of the fish in nature lose their melanophores and thereby become gold-coloured. Before metamorphosis, all fish look alike. We asked whether the pre-transformed offspring of gold and normal morphs might differ in their morphological and physiological responses to white and to black backgrounds despite looking the same. More chromatophores developed in all juveniles exposed for two weeks to a black than to a white background. When moved from one background to the other, the fish adjusted to white faster than to black. Most importantly, genetically normal fish (N) colour-matched quicker than genetically gold fish (G) irrespective of the direction of change. Adjustment in G was also more variable, indicating poorer control. A parallel pattern was recorded photometrically in melanophores of excised scales: aggregation of pigment was faster than dispersion. And pigment in the melanophores of N aggregated and dispersed swifter than did those of G. Therefore, pre-metamorphosed juvenile G could be more vulnerable to predation than are juvenile N. This reveals a possible source of selection against the offspring of gold morphs before they become gold-coloured.     

Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe

Abstract A repeated DNA sequence was isolated from Fusobacterium necrophorum biotype AB, strain FnS1. The repeated sequence shared considerable homology with the transposase gene from the Pseudomonas syringiae insertion sequence IS801. The repeat sequence was used together with a 16S ribosomal RNA gene probe to type F. necrophorum isolates using restriction fragment length polymorphisms. The probes revealed differences between several clinical isolates and will be useful tools to study the epidemology of ovine foot abscess and other diseases caused by F. necrophorum .     

Expressed hypervariable polymorphism of apolipoprotein (a)

Elevated plasma lipoprotein (a) (LP(a] levels are an independent predictor of the development of premature atherosclerosis in humans. The LP(a) particle consists of two disulfide-linked proteins, apolipoprotein (APO) B and APO(a). The APO(a) is a highly glycosylated protein which carries the LP(a) antigen. Genetic polymorphism in the APO(a) molecule has been reported, and, depending on the sensitivity of the method used, 6-11 alleles at the APO(a) structural locus have been documented in the literature. In this investigation, we have used a high-resolution SDS-agarose electrophoresis method followed by immunoblotting to screen APO(a) polymorphism in 54 families with 130 offspring. This method identified a total of 23 different APO(a) isoforms, and their genetic basis was confirmed in families. In addition to the detectable products of 23 APO(a) alleles, the family data predict the existence of a "null" allele. Of the total 270 individuals tested, 209 (77.4%) revealed double-banded phenotypes and 61 (22.6%) revealed single-banded phenotypes. In the unrelated sample of 140 individuals, however, 114 (81.4%) and 26 (18.6%) had double- and single-banded phenotypes, respectively. When the segregation pattern of single-banded phenotypes in the unrelated sample was followed in families, only nine (6.4%) were found to be true homozygotes, and the remaining 17 (12.2%) were classified as heterozygotes for the null allele. Of the 276 possible phenotypes predicted for 23 alleles in a large population, we observed 115 (42%) phenotypes in our restricted sample. On the basis of our results from the family data, we hypothesize the existence of at least 24 alleles, including a null allele, at the APO(a) structural locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel cryptic plasmid, pHLHK26, in Laribacter hongkongensis

We report the complete nucleotide sequence and characterization of a cryptic plasmid, pHLHK26, recovered from a strain of Laribacter hongkongensis isolated from a patient with community acquired gastroenteritis. pHLHK26 consists of 8700 bp, with G + C content 51.3%. The copy number (mean +/- SD) is 0.57 +/- 0.07 and it is stable after four passages (about 240 generations) in the absence of selection. There is a predicted origin of replication that consists of a DnaA box and five 22-bp direct repeats. pHLHK26 has four ORFs with two genes encoded in the sense direction and the other two in antisense direction. These four ORFs encode a putative plasmid partitioning protein of the ParA family, a putative protein that contains putative ADP-ribose 1"-phosphatase activity belonging to the Appr-1-p processing enzyme family, a putative recombinase (TniR) of the resolvase/invertase family, and a putative replication protein, respectively. We speculate that pHLHK26 is a theta, possibly Class A, replicative plasmid, as it contains an origin of replication with AT-rich region, a number of iterons and a DnaA box and a gene that encodes a replicative protein most homologous to those of other theta replicative plasmids and it shares eight of the nine positions of the consensus sequence TTAT(C/A)CA(C/A)A (TTTTCCACA in pHLHK26) in the DnaA boxes observed in other classical examples of Class A plasmids of this group.