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PubMed2007

No evidence for an mtDNA role in sperm motility: data from complete sequencing of asthenozoospermic males.

The first complete mitochondrial DNA (mtDNA) sequences (approximately 16,569 bp) in 20 patients with asthenozoospermia and a comparison with 23 new complete mtDNA sequences in teratoasthenozoospermic individuals, confirmed no sharing of specific polymorphisms or specific mitochondrial lineages between these individuals. This is strong evidence against the accepted claim of a major role played by mtDNA in male fertility, once supported by haplogroup association studies based on the screening of hypervariable region I. The hypothesis of maternally driven selection acting in male reproductive success must thus be treated with caution.

Analysis of Variance↗
PubMed1988

A large deletion encompassing the entire alpha-like globin gene cluster in a family of northern European extraction.

We describe a new deletional form of alpha thalassemia segregating in three generations of a family of northern European origin. A full-term female girl had hypochromic, microcytic anemia since early infancy associated with delayed language development, slow growth and weight gain. Hematologic studies suggested the presence of alpha thalassemia. Gene-blotting studies showed no abnormal alpha-like globin gene fragments; however, studies of inheritance of informative polymorphic restriction fragments using zeta, alpha and 3'-alpha-hypervariable region (3'-HVR) probes showed evidence for an extensive deletion encompassing the entire alpha-like globin gene cluster. The 3' breakpoint of this deletion maps beyond the 3'-HVR, a region implicated as a hot spot for the generation of other large deletional events within the alpha-like cluster. The 5' breakpoint maps at least 10 kilobases (kb) 5' to the zeta-globin gene. The minimum size estimate for this deletion is greater than 47 kilobases.

Child, Preschool↗
PubMed1989

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.

Cloning, Molecular↗
PubMed1990

The human minisatellite consensus at breakpoints of oncogene translocations.

A reexamination of human minisatellite (hypervariable) regions following the cloning and sequencing of the new minisatellite, VTR1.1, revealed that many of these structures possessed a strongly conserved copy of the chi-like octamer, GC[A/T]GG[A/T]GG. In oncogene translocations apparently created by aberrant VDJ recombinase activity, this VTR octamer was often found within a few bases of the breakpoint (p less than 10(-10)). Three bcl2 rearrangements which occurred within 2 bp of one another were located precisely adjacent to this consensus; it defined the 5' border of that oncogene's major breakpoint cluster. Several c-myc translocations also occurred within 2 bp of this sequence. While the appearance of a chi-like element in polymorphic minisatellite sequences is consistent with a role promoting either recombination or replication slippage, the existence of such elements at sites of somatic translocations suggests chi function in site-specific recombination, perhaps as a subsidiary recognition signal in immunoglobulin gene rearrangement. We discuss the implications of these observations for mechanisms by which oncogene translocations and minisatellite sequences are generated.

Base Sequence↗
PubMed1994

Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis.

The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus.

Base Composition↗
PubMed1996

Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro.

We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The resulting lacZalpha peptide fusion protein remained functional. Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation. With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis. Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations. DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis. RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum. The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites. Results indicate that recombination is another source of mutations during viral replication.

Base Sequence↗
PubMed1981

The nucleotide sequence of the large ribosomal RNA gene and the adjacent tRNA genes from rat mitochondria.

We have sequenced the Eco R(1) fragment D from rat mitochondrial DNA. It contains one third of the tRNA (Val) gene (the remaining part has been sequenced from the 3' end of the Eco R(1) fragment A) the complete gene for the large mt 16S rRNA, the tRNA (Leu) gene and the 5' end of an unidentified reading frame. The mt gene for the large rRNA from rat has been aligned with the homologous genes from mouse and human using graphic computer programs. Hypervariable regions at the center of the molecule and highly conserved regions toward the 3' end have been detected. The mt gene for tRNA Leu is of the conventional type and its primary structure is highly conserved among mammals. The mt gene for tRNA(Val) shows characteristics similar to those of other mt tRNA genes but the degree of homology is lower. Comparative studies confirm that AGA and AGG are read as stop codons in mammalian mitochondria.

Animals↗
PubMed2005

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus.

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At approximately 2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum.

Amino Acid Sequence↗
PubMed1995

Genetic epidemiology of rheumatoid disease.

The aetiology of rheumatoid arthritis (RA) has both genetic and environmental origins. However, the relationship of these with each other and with RA disease is complicated as both genes and environment may be protective or render individuals susceptible. Diagnostic certainty and disease heterogeneity have posed serious hurdles for investigating RA. Patients with similar disease phenotypes may have different aetiologies and it may now be more appropriate to investigate genetic susceptibility with respect to disease process or characteristic immunopathology. Disease concordance studies have been a classical approach to estimating the upper threshold of the genetic contribution to RA and recent studies have approximately halved the much quoted figure of 32% reported by Lawrence for RA concordance in monozygotic twins. However, the interpretation of twin data has to be treated with caution as MZ twins can differ in terms of somatic rearrangement/mutation of immunoglobulin and T cell receptor genes, X chromosome inactivation in females, genomic imprinting and in utero immune relationship. Calculations based on heritability put the genetic component in RA much higher. HLA is an important genetic factor in RA and risk is thought to be associated with a consensus sequence of amino acids within the third hypervariable region of certain DRB1 alleles. It is now clear that HLA is not particularly associated with onset of synovitis but is more associated with progression and severity of the disease process. The HLA-DRB1*0401/0404 genotype is particularly associated with severe, erosive and seropositive RA. Clear differences in RA susceptibility exist between males and females and this may be attributed to hormonal status. Males have a higher threshold requirement for developing RA and this may explain why RA in males is more associated with HLA risk alleles. We have now reached a critical time for the investigation of RA aetio-pathology. Recent advances in molecular biology and automated microsatellite gene scanning technology are making it possible to map disease susceptibility genes over the whole genome in common disorders such as RA. This will require large numbers of well characterised multiplex affected families. It is anticipated that some of the genes identified will fit in with our current concepts of which inflammatory and immune processes are pathologically important in RA. However others may be a major surprise.

Arthritis, Rheumatoid↗
PubMed1983

Isoelectric focusing of the inbred mouse antibody to bacterial alpha-amylase.

In the IgG antibody response to bacterial alpha-amylase (B alpha A) assayed by the enzymatic procedure, C3H/He (C3) mice were high and C57BL/6 (B6) mice were low responders. High responsiveness was inherited as a dominant characteristic in (B6XC3)F1 hybrid mice. In these strains, the primary antibody response was analyzed for heterogeneity by isoelectric focusing (IEF). The IEF spectra were visualized with the use of the capacity of antibody to inhibit the amylase activity of antigen. Increases in the antigen dose and in the time interval between immunization and bleeding resulted in increases in antibody titers accompanied by strong staining of focused antibodies and by the expansion of the pH range where antibodies were focused. High responsiveness in C3 and F1 hybrid mice was also associated with the increase in intensity of stain and the rapid expansion of pH range of focused antibodies. Another strain difference was noted in the isoelectric point (pI) values of antibodies taken early in the primary response. B6 antisera contained those fractions of antibodies focusing over a more alkaline area than C3 antibodies. A similar strain difference in the pI values of antibodies occurred in the response to an irrelevant antigen, Taka-amylase A (TAA), suggesting that the hypervariable regions of antibody molecules play no major part in the strain difference observed. Antisera from F1 hybrid mice displayed bands covering the combined pH ranges of B6 and C3 spectrotypes.

Animals↗
PubMed1997

The geographic apportionment of mitochondrial genetic diversity in east African chimpanzees, Pan troglodytes schweinfurthii.

This study is a geographically systematic genetic survey of the easternmost subspecies of chimpanzee, Pan troglodytes schweinfurthii. DNA was noninvasively collected in the form of shed hair from chimpanzees of known origin in Uganda, Rwanda, Tanzania, and Zaïre. Two hundred sixty-two DNA sequences from hypervariable region 1 of which of the mitochondrial control region were generated. Eastern chimpanzees display levels of mitochondrial genetic variation which are low and which are similar to levels observed in humans (Homo sapiens). Also like humans, between 80% and 90% of the genetic variability within the eastern chimpanzees is apportioned within populations. Spatial autocorrelation analysis shows that genetic similarity between eastern chimpanzees decreases clinically with distance, in a pattern remarkably similar to one seen for humans separated by equivalent geographic distances. Eastern chimpanzee mismatch distributions (frequency distributions of pairwise genetic differences between individuals) are similar in shape to those for humans, implying similar population histories of recent demographic expansion. The overall pattern of genetic variability in eastern chimpanzees is consistent with the hypothesis that the subject has responded demographically to paleoclimatically driven changes in the distribution of eastern African forests during the recent Pleistocene.

Africa, Eastern↗
PubMed1994

Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.

We cloned 17 different PCR fragments encoding VH genes of camel (Camelus dromedarius). These clones were derived from the camel heavy chain immunoglobulins lacking the light chain counterpart of normal immunoglobulins. Insight into the camel VH sequences and structure may help the development of single domain antibodies. The most remarkable difference in the camel VH, consistent with the absence of the VL interaction, is the substitution of the conserved Leu45 by an Arg or Cys. Another noteworthy substitution is the Leu11 to Ser. This amino acid normally interacts with the CH1 domain, a domain missing in the camel heavy chain immunoglobulins. The nature of these substitutions agrees with the increased solubility behavior of an isolated camel VH domain. The VH domains of the camels are also characterized by a long CDR3, possibly compensating for the absence of the VL contacts with the antigen. The CDR3 lacks the salt bridge between Arg94 and Asp101. However, the frequent occurrence of additional Cys residues in both the CDR1 and CDR3 might lead to the formation of a second internal disulfide bridge, thereby stabilizing the CDR structure as in the DAW antibody. Within CDRs of the camel VH domains we observe a broad size distribution and a different amino acid pattern compared with the mouse or human VH. Therefore the camel hypervariable regions might adopt structures which differ substantially from the known canonical structures, thereby increasing the repertoire of the camel antigen binding sites within a VH.

Amino Acid Sequence↗
PubMed2002

Single-strand conformation polymorphism (SSCP) assays for major histocompatibility complex B genotyping in chickens.

We have developed a DNA-based method for defining MHC B system genotypes in chickens. Genotyping by this method requires neither prior determination of allele-specific differences in nucleotide sequence nor the preparation of haplotype-specific alloantisera. Allelic differences at chicken B-F (class I) and B-L (class II) loci are detected in PCR single-strand conformation polymorphism (SSCP) assays. PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the two class I and two class I loci of the B-complex and used to generate DNA fragments that are heat- and formamide-denatured and then analyzed on nondenaturing polyacrylamide gels. PCR primer pairs were tested for the capacity to produce SSCP patterns allowing the seven B haplotypes in the MHC B congenic lines, and seven B haplotypes known to be segregating in two commercial broiler breeder lines to be distinguished. Primer pairs were further evaluated for their capacity to reveal the segregation of B haplotypes in a fully pedigreed family and in a closed population. Concordance was found between SSCP patterns and previously assigned MHC types. B-F and B-L SSCP patterns segregated in linkage as expected for these closely linked loci. We conclude that this method is valuable for defining MHC B haplotypes and for detecting potential recombinant haplotypes especially when used in combination with B-G (class IV) typing by restriction fragment pattern.

Animals↗
PubMed1991

Limited heterogeneity of the HLA class II contribution to susceptibility to rheumatoid arthritis is suggested by positive associations with HLA-DR4, DR1 and DRw10.

Most patients with rheumatoid arthritis (RA) exhibit an HLA class II epitope found on most DR4 and DR1 molecules. We have investigated the possibility of other associations being present in the minority of patients lacking these antigens. One hundred and eighty patients with classical or definite RA and 100 controls were assigned HLA-DR, DQ and DP types by a DNA-based system using sequence-specific oligonucleotides to probe amplified class II alleles amplified by the polymerase chain reaction. The expected associations with DR4 (relative risk 10, P less than 0.0001) and DR1 (relative risk 2.3, P less than 0.001) were observed and only 13% of individuals lacked both of these alleles compared with 54% of age- and race-matched controls. A significant association with DRw10 (P = 0.02) was also observed in the DR4/DR1-negative RA group (3/23 patients compared with 0/54 DR4/DR1-negative controls). No novel associations with other DR, DQ, or DP alleles were evident in contrast to some previous studies. The third allelic hypervariable region of the DR beta chain of DRw10 contains a similar amino acid sequence to that found in many DR4 and DR1 molecules. These results extend this correlation and suggest that this susceptibility determinant may account for the HLA-linked susceptibility in 89% of our cases of RA.

Amino Acid Sequence↗
PubMed1993

The association of HLA DR beta alleles with self-limiting and persistent forms of early symmetrical polyarthritis.

RA is associated with a group of class II DR beta alleles, which share a conserved sequence in the third allelic hypervariable region (3AHVR). These include the DR4 subtypes Dw4, Dw14 and Dw15, and also DR1. In contrast Dw10 and Dw13 which are also DR4 subtypes show no association with RA and have a quite distinct 3AHVR. We have assessed the frequency of these alleles in 55 patients who initially presented with symmetrical peripheral polyarthritis suggestive of RA. After 4 years 27 had progressed to definite or classical RA, while 28 had never fulfilled the criteria for this disease. Dw4 was markedly elevated in the rheumatoid group (17/23) compared with either the non-rheumatoids (5/28) or healthy controls (12/100). Dw14 and non-DR4 associated DR1 were not elevated in either group of patients. This study suggests that the DR beta association with RA is likely to facilitate persistence or severity of disease rather than the initial induction of symmetrical peripheral arthritis.

Adult↗
PubMed1994

HLA class 2 genes in Singaporean Chinese rheumatoid arthritis.

This study analysed HLA class 1 and 2 allele associations in Singaporean Chinese patients with RA. Seventy patients (ARA definite or classical) and 80 controls were typed for HLA class 1 alleles by serology and class 2 alleles by serology and the PCR/SSO method. RA patients had higher frequencies of DRB1*0405 (40 vs 12.5%; corrected probability value (PC) < 0.02, relative risk (RR) = 4.7, 95% confidence limit (CL) 2.1-10.6), DRB1*1001 (14.3 vs 1.3%; PC = 0.06, RR = 13.2, 95% CL 1.6-105.7), DQB1*0401 (38.6 vs 12.5%; P = 0.006, RR = 4.4, 95% CL 1.9-10.0) and DQB1*0501 (20 vs 5%; PC = 0.048, RR = 4.8, 95% CL 1.5-15.2). It is concluded that Chinese RA is associated primarily with HLA DRB1*0405 and DRB1*1001 which share common amino acid sequences in the third hypervariable region of the DR beta chains shown to be associated with RA in other ethnic groups. Patients without DRB1*0405 and *1001 had a higher frequency of DRB1*0901, which is in linkage disequilibrium with HLA B46 in the Chinese.

Adolescent↗
PubMed1996

Polymyalgia rheumatica is associated with both HLA-DRB1*0401 and DRB1*0404.

The frequency of HLA-DRB1 alleles was determined in 68 Caucasoid patients with polymyalgia rheumatica (PMR) and 140 controls using polymerase chain reaction (PCR) sequence-specific oligonucleotide typing. In keeping with previous studies, an increased frequency of DRB1*04 was observed in patients [55.9% vs 35.0%, odds ratio (OR) 2.4, 95% confidence interval (CI) 1.3-4.4]. HLA-DRB1*0101 frequency was also increased in patients, although less confidence could be placed on this association (19.1% vs 14.3%, OR 1.4, 95% CI 0.6-3.3). HLA-DRB1*04 subtyping indicated that the frequencies of both DRB1*0401 (38.2% vs 22.1%, OR 2.2, 95% CI 1.0-4.3) and DRB1*0404 (16.2% vs 5.0%, OR 3.7, 95% CI 1.2-11.1) were specifically raised. An increased frequency of the RA shared epitope (QKRAA/QRRAA) was also observed in this group (75.0% vs 44.2%, OR 3.8, 95% CI 1.9-7.6). When the analysis was restricted to only DRB1*04-negative patients and controls, the frequencies of DRB1*0301, *11 and *08 were marginally raised. However, no obvious relationship appeared to exist between PMR susceptibility and DRB1 alleles carrying the DYF conserved epitope in the second hypervariable region. Autoantibodies to thyroid antigens were present in 23% of patients. An increased frequency of DRB1*0301 was observed in patients with thyroid microsomal antibodies compared to those without (54.5% vs 24.6%, OR 3.7, 95% CI 0.8-17.0). This increase was not observed in patients with thyroglobulin autoantibodies. These data indicate that both DRB1*0401 and *0404 are associated with PMR, and that this may extend to include DRB1*0101. The immunogenetic profile of susceptibility markers in this condition appears to be similar to that in rheumatoid arthritis.

Aged↗
PubMed1997

HLA DRB1* typing and cartilage oligomeric matrix protein (COMP) as predictors of joint destruction in recent-onset rheumatoid arthritis.

The carriage of a characteristic sequence of amino acids at position 67-72 in the third hypervariable region of the HLA DRB1 chain has been linked to susceptibility to rheumatoid arthritis (RA). Whether this epitope is also a predictor of more severe disease remains controversial. Cartilage oligomeric matrix protein (COMP) is a protein, the serum levels of which have been found to correlate with large joint destructive disease in previous work. In this paper, we compare DRB1* typing and serum COMP levels in a prospectively observed group of RA patients with or without early hip joint destruction. The COMP levels at study inclusion, median 11 months from onset of symptoms, were significantly higher in the patients with early hip joint destruction compared to the patients in the more benign group. There was no difference in the number of disease susceptibility-related epitopes between the groups. DRB1*04, in contrast, was found among 8/8 patients with hip destruction, but also in 5/8 more benign cases. We conclude that in this type of RA patient, COMP serum levels are more informative predictors of aggressive disease than HLA DRB1* typing.

Alleles↗