Immunogenetic studies of rhesus monkeys. 8. A new reagent of the G blood group system and an example of the founder principle

A new reagent called G-3' has been discovered which detects the product of the G ³allele, regardless of the other G allele in the genotype. With this new reagent we can delineate nine of 10 genotypes of the G blood group system. The explicit detection of G ³revealed an example of the founder principle in that the frequency of this allele between the feral and laboratory-reared monkeys was significantly different.     

A structural model of 5S RNA from E. coli based on intramolecular crosslinking evidence.

We describe new results obtained using the bifunctional chemical reagent phenyldiglyoxal (PDG) to study the intramolecular crosslinking of ribosomal 5S RNA from E. coli. In a previous publication (Wagner & Garrett [1]) we reported the identification of a crosslink in the stem region of 5S RNA (G2-G112) using the same reagent but were unable to obtain further information because of the presence of monofunctional adducts which confused the analyses. To overcome this problem, we have removed the monoaddition products by coupling them via their free reagent ends to a solid support bearing reactive groups. Using this system we have been able to identify a new crosslink G41-G72 in native 5S RNA which has considerable structural implications. We propose a structural model in which the proximity of both nucleotides is maintained by secondary interactions.     

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians.     

The proximity of the C-terminal domain of Escherichia coli ribosomal protein L7/L12 to L10 determined by cysteine site-directed mutagenesis and protein-protein cross-linking.

Oligonucleotide-directed mutagenesis was used to produce a serine 89 to cysteine 89 substitution in the C-terminal globular domain of Escherichia coli ribosomal protein L7/L12. Cys-89 represented the only cysteine residue in the protein. L7/L12Cys89 was overproduced in E. coli and purified. An allele replacement strain was also constructed. Growth of this strain was indistinguishable from that of wild type. Ribosomes from the allele replacement strain were used to determine the location of the C-terminal domains of L7/L12 by disulfide cross-linking. A new homobifunctional cysteine-specific cross-linking reagent, 1,4-di[3'-(2'-pyridyldithio)-propionamido]butane, and diagonal gel electrophoresis were used to identify ribosomal proteins cross-linked to L7/L12Cys89. A cross-link between L7/L12 and the single cysteine in L10 was found, in addition to L7/L12 dimers. The L7/L12Cys89-L10 cross-link locates the C-terminal domain of at least one L7/L12 dimer on the body of the large subunit and supports our previous model (Olson, H. M., Sommer, A., Tewari, D. S., Traut, R. R., and Glitz, D. G. (1986) J. Biol. Chem. 261, 6924-6932) that depicts one of the two dimers of L7/L12 on the surface of the body of the 50 S subunit in a bent conformation with the C-terminal domain in close proximity to the N-terminal domain at the base of the stalk.     

Carbodiimide modification analysis of aminoacylated yeast phenylalanine tRNA: evidence for change in the apex region.

The G- and U-specific reagent, carbodiimide was used to probe the solution structure of aminoacylated yeast phenylalanine tRNA. Both quantitative and qualitative changes in modification were observed when the modification patterns of tRNA-CCA(3'OH), tRNA-CCA(3'NH2) and phe-tRNA-CCA(3'NH2) were compared. Five nucleotides were modified in all cases, D16 and G20 in the D-loop, U33 and Gm34 in the anticodon loop and U47, in the region of the extra arm. Small changes occurred in the D-loop with incorporation of the adenosine analogue manifest as new, low levels of modification of G22 (D-stem) and a loss of sensitivity to Mg+2 in modification of D16. Aminoacylation resulted in new modification of G19, modification of a residue in the T psi CG sequence, and a 2.5-fold increase in modification of G22. Taken together the results show that aminoacylation causes increased exposure of bases in the apex region of the L-shaped molecule where the D- and psi-loops are joined. The effects observed could occur as a consequence of stable or dynamic changes in conformation.     

A single nucleotide polymorphism in the matrix metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes.

Interstitial collagen gives fetal membranes tensile strength, and membrane rupture has been attributed to collagen degradation. A polymorphism at -1607 in the matrix metalloproteinase-1 (MMP-1) promoter (an insertion of a guanine (G)) creates a core Ets binding site and increases promoter activity. We investigated whether this polymorphism is functionally significant for MMP-1 expression in amnion cells and whether it is associated with preterm premature rupture of the membranes (PPROM). The 2G promoter had >2-fold greater activity than the 1G allele in amnion mesenchymal cells and WISH amnion cells. Phorbol 12-myristate 13-acetate (PMA) increased mesenchymal cell nuclear protein binding with greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was significantly greater in cells with a 1G/2G or 2G/2G genotype compared with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells. A significant association was found between fetal carriage of a 2G allele and PPROM. We conclude that the 2G allele has stronger promoter activity in amnion cells, that it confers increased responsiveness of amnion cells to stimuli that induce MMP-1, and that this polymorphism contributes to the risk of PPROM.     

Assessment of the FAM174A 11G allele as a risk allele for equine metabolic syndrome

An 11G nucleotide repeat in the 3′ UTR of FAM174A was recently postulated as a risk allele with a dominant mode of inheritance for equine metabolic syndrome (EMS) and laminitis status in Arabian horses. The objective of this project was to evaluate this hypothesis in a large and diverse across-breed population. A total of 301 ponies, 292 Morgans, 64 Arabians, 49 Tennessee Walking Horses and 59 Quarter Horses were genotyped for six observed G repeat alleles in the FAM174A 3′ UTR. Phenotype data included laminitis status, baseline insulin, glucose, non-esterified fatty acids, triglycerides, adiponectin, leptin, ACTH, insulin and glucose post oral sugar test, and two proxies for insulin resistance. The 11G allele frequencies were 18.8, 6.9, 1.8, 0.2 and 0.0% in the Arabians, Tennessee Walkers, ponies, Morgans and Quarter Horses respectively. Association analyses between FAM174A genotype and EMS phenotypes, and between allele count and EMS phenotypes, identified no statistically significant associations. When a dominant effect for the 11G allele was evaluated, a statistically significant association with adiponectin levels was identified in the ponies, and pairwise comparisons revealed that the estimated marginal means were higher in ponies with the 11G allele vs. alternative alleles (i.e. the allele had a protective effect). In conclusion, our data do not support the FAM174A 11G allele as a risk allele for EMS in our studied breeds.     

PGI*3(Israel), a new,unstable allele in the phosphoglucose isomerase system

Summary Phosphoglucose isomerase (PGI) and 16 other biochemical genetic markers were studied in an Israeli-Arab family previously described for hereditary deficiency of adenylate kinase (AK) and glucose 6-phosphate dehydrogenase (G6PD). In this inbred family a rare PGI*3 allele was observed in 11 of 32 members tested, indicating an autosomal codominant inberitance. The electrophoretic mobility of this allele is similar to that of the PGI*3 allele found in Indian populations, but unlike the Indian allele, it has a very low specific activity and heat stability. This PGI*3 allele, designated PGI*3 (Israel), seems to be a different unstable mutation and along with AK and G6PD deficiencies seems to be associated with severe anaemia.     

Further characterization of the G blood group system of rhesus monkeys

We describe a new antiserum that has the unique ability to distinguish homozygous from heterozygous genotypes in the G blood group system of rhesus monkeys. With this new typing serum (reagent), all 10 possible genotypes in this system can be distinguished and the utility of blood typing for genetic studies has been greatly increased.     

Role of Progesterone Receptor Polymorphisms in the Recurrent Spontaneous Abortions: Indian Case

Background

We attempt to ascertain if the 3 linked single nucleotide polymorphisms (SNPs) of the Progesterone Receptor (PR) gene (exon 1: G 1031 C; S344T, exon 4: G 1978 T; L660V and exon 5: C 2310 T; H770H) and the PROGINS insertion in the intron G, between exons 7 and 8, are associated with Recurrent Spontaneous Abortion (RSA) in the Indian population.

Methodology/Principal Findings

A total of 143 women with RSA and 150 controls were sequenced for all the 8 exons looking for the above 3 linked SNPs of the PR gene earlier implicated in the RSA, as well as for any new SNPs that may be possibly found in the Indian population. PROGINS insertion was screened by electrophoresis. We did not find any new mutations, not observed earlier, in our population. Further, we did not find significant role of the *2 allele (representing the mutant allele at the three SNP loci) or the T2 allele (PROGINS insertion) in the manifestation of RSA. We also did not find an LD pattern between each of the 3 SNPs and the PROGINS insertion.

Conclusions/Significance

The results suggest that the PR gene mutations may not play any exclusive role in the manifestation of RSA, and instead, given significantly higher frequency of the *2 allele among the normal women, we surmise if it does not really confer a protective role among the Indian populations, albeit further studies are required in the heterogeneous populations of this region before making any conclusive statement.     

汉族人群NOS3 A-922G、NOS3 T-786C 与NOS3 G894T SNP的等位基因及其组合分布与高血压的相关性

为了分析汉族人群一氧化氮合酶基因NOS3 A-922G、NOS3 T-786C 与NOS3 G894T单核苷酸多态性（single nucleotide polymorphism,SNP）的等位基因及其组合分布与高血压病的相关性,选取无亲缘关系的高血压病人192例(男97例,女95例)以及无亲缘关系的健康个体122例（男76例,女46例）为对照组,提取静脉血白细胞基因组DNA,采用等位基因特异性引物PCR技术检测NOS3 A-922G、NOS3 T-786C 与NOS3 G894T 3个位点的基因型。其结果显示：高血压病组与对照组NOS3 G894T、NOS3A-922G及NOS3 T-786C各等位基因型及其基因单倍型频率比较无显著性差异(P>0.05)。男、女性别分层研究：无论男亚组还是女亚组均未发现NOS3 A-922G、NOS3 T-786C 与NOS3 G894T各个位点SNP与高血压病有相关性。等位基因组合分布研究发现NOS3 G894G +A-922G+T-786T组合基因型总体频率分布在高血压病组与正常对照组之间有显著性差异(P<0.05,χ2= 4.5944)。男、女性别分层研究：男亚组上述3个位点SNP的各个组合基因型分布频率在高血压病组与正常对照组之间无显著性差异(P>0.05);女亚组中携带NOS3 G894G+A-922G+T-786C 的组合基因型分布频率在高血压病组与正常对照组之间有显著性差异(P<0.01,χ2=8.502)。研究发现,在中国汉族人群中NOS3A–922 G、NOS3 T-786C 与NOS3 G894T SNP与高血压病无明确的相关性,且无性别差异。组合分布研究发现,NOS3 G894G+A-922G+T-786C 的组合基因型分布频率在高血压病女性亚组较健康女性亚组明显减低,提示携带该组合基因型女性人群可能不易患高血压病。     

两个国内小型猪品种GH基因部分序列的多态性分析

目的对西藏小型猪和广西巴马小型猪生长激素基因（GH基因）部分序列的多态性进行分析。方法采用内切酶ApaI和Hin6I对西藏小型猪（108头）和广西巴马小型猪（132头）GH基因-119 bp～＋486 bp之间的区域进行PCR-RFLPs分析。结果 （1）从ApaI酶切产生的结果来看,ApaI酶切产生A（449 bp＋101 bp＋55bp）,B（316 bp＋133 bp＋101 bp＋55 bp）两种等位基因。等位基因A的频率高于等位基因B,等位基因A为优势基因。AA基因型频率高于AB和BB基因型频率;（2）从Hin6I酶切产生的结果来看,Hin6I酶切产生G1（605bp）、G2（498 bp＋107 bp）、G3（449 bp＋156 bp）、G4（449 bp＋107 bp＋49 bp）四种等位基因。等位基因G4的频率高于等位基因G1、G2、G3。等位基因G1频率很低。基因型G2G3、G2G4、G3G4、G4G4的频率较高。（3）由酶切产生的基因和基因型多态性,发现西藏小型猪与广西巴马小型猪在该基因部分序列差异不显著（P〉0.05）。结论国内的优质实验用小型猪,如西藏小型猪和广西巴马小型猪等位基因A频率均较高。     

The occurrence of the alpha G-Philadelphia-globin allele on a double-locus chromosome.

Hb G-Philadelphia, an alpha-globin allele, is expressed as either 20%, 30%, or 40% of the total hemoglobin. Restriction analyses published thus far have shown that among persons with 30% and 40% hemoglobin (Hb) G the alpha G allele is seen only in a single-locus haplotype. We now report the identification of a second haplotype in which the alpha G allele is found in tandem with an alpha A allele. This haplotype has been found present in DNA from the members of one family in which Hb G is expressed as 20% of the total hemoglobin, determined by both cellulose acetate electrophoresis and high-performance liquid chromatography (HPLC). Synthesis was balanced in all individuals. The identification of a variant alpha-globin allele in two distinct haplotypes presents the possibility of independent mutation. However, an alternative explanation cannot be ruled out; namely, that the original allele may have become distributed among the two haplotypes by unequal crossing-over.     

A single nucleotide polymorphism in 3'-untranslated region contributes to the regulation of Toll-like receptor 4 translation

We have previously shown that a single nucleotide polymorphism rs11536889 in the 3'-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK(4) (Pam(3)CSK(4)), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3'-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.     

Catalytic site-specific cleavage of a DNA-target by an oligonucleotide carrying bleomycin A5.

Oligonucleotide reagents have been created which are capable of catalytic site-specific cleavage of DNA-targets. The oligonucleotide reagent Blm-R-pd(CCAAACA) bearing the bleomycin A5 (Blm-RH) residue was used to degrade the DNA-target pd(TGTTTGGCGAAGGA). It has been shown that at equimolar reagent: target concentration the bleomycin oligonucleotide derivative can repeatedly cleave the target at G9, G7, T5, T4 and T3 in site-specific manner. This paper demonstrates that with a 10-fold excess of the DNA-target relative to the reagent 30% degradation of the target was observed primarily at a single position G7. The paper also shows that one reagent molecule containing bleomycin A5 residue was capable to degrade three molecules of the DNA-target. The catalytic activity of Blm-R-pd(CCAAACA) was the highest in the temperature range close to the melting temperature of the reagent-target complex, that is under conditions where the oligonucleotide reagent can form a complementary complex and easily dissociate to interact with the next molecule of the target. The number of target molecules degraded by the bleomycin reagent is limited by the degradation of the antibiotic residue itself.     

ABCA1 single nucleotide polymorphisms on high-density lipoprotein-cholesterol and overweight: the D.E.S.I.R. study

The adenosine triphosphate-binding cassette A1 (ABCA1) gene plays a key role in reverse cholesterol transport. Some ABCA1 gene polymorphisms have been associated with high-density lipoprotein-cholesterol (HDL-C) concentrations. The aim of this study was to assess the effect of three polymorphisms, C69T, G378C, and G1051A (R219K), on HDL-C levels and their interaction with BMI in more than 5000 French whites from the D.E.S.I.R. (Data from an Epidemiological Study on the Insulin Resistance syndrome) cohort study. The T allele of the C69T single nucleotide polymorphism (SNP) was associated with higher HDL-C levels in normal-weight men (BMI <25 kg/m(2)). The C allele of the G378C SNP was associated with lower HDL-C in overweight subjects (BMI > or =25 kg/m(2)). For the G1051A SNP, in the normal-weight group, the minor A allele was significantly associated with higher HDL-C levels. In contrast, in overweight people, the minor allele was associated with lower HDL-C levels. After accounting for multiple testing, empiric p values remained significant for the associations between G378C SNP and HDL-C in the overweight group and between G1051A SNP and HDL-C in the normal-weight group. This study suggests that ABCA1 gene polymorphisms modulate HDL-C concentrations, in interaction with BMI, and, thus, they might influence cardiovascular risk in the general population.     

Characterization of two new point mutations in the low density lipoprotein receptor genes of an English patient with homozygous familial hypercholesterolemia.

Two new point mutations have been detected in the low density lipoprotein (LDL) receptor gene of a patient with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The patient is a compound heterozygote, in whom the mutant allele inherited from his English father has a single base substitution of A for G in exon 3, changing the codon for residue 80 in the mature protein from glutamic acid to lysine. The mutant allele inherited from his mother, who is of Irish origin, has a single base pair deletion in the codon for residue 743 in exon 15 that causes a frameshift and introduces a new stop codon in the adjacent position. The glu80 to lys mutation results in a transport-defective phenotype and a mature protein that migrates abnormally slowly on nonreduced SDS-PAGE, but normally under reducing conditions; this was confirmed by site-directed mutagenesis and expression in vitro. The deletion in exon 15 results in a null phenotype in which the putative truncated receptor protein cannot be detected in cultured skin fibroblasts and the amount of mRNA derived from the allele is reduced. The glu80 to lys mutation was found in a further five unrelated individuals in a sample of 200 FH patients from the London area and in 11 from a sample of 77 FH patients from Manchester. Haplotype analysis suggested that all the patients had inherited this allele from a common ancestor. The deletion in exon 15 was not found in the London sample, nor in any unrelated individuals in the Manchester sample.     

Polymorphisms in the Regulatory Region of the Cyclophilin A Gene Influence the Susceptibility for HIV-1 Infection

Background

Previous studies have demonstrated an association between polymorphisms in the regulatory regions of Cyclophilin A (CypA) and susceptibility to both HIV-1 infection and disease progression. Here we studied whether these polymorphisms are associated with susceptibility to HIV-1 infection and disease progression in the Amsterdam Cohort on HIV-1 infection and AIDS (ACS) in a group of men having sex with men (MSM) and drug users (DU).

Methodology/Principal Findings

We screened participants of the ACS for the C1604G and A1650G polymorphisms in the regulatory regions of CypA. The prevalence of the 1650G allele was significantly higher in high risk seronegative MSM than in HIV-1 infected MSM. However, C1604G or A1650G were not associated with the clinical course of infection in MSM of the ACS. Interestingly, participants of the ACS-DU who carried the 1604G allele showed a significantly accelerated progression when viral RNA load above 10^4.5 copies per ml plasma was used as an endpoint in survival analysis.

Conclusion/Significance

The results obtained in this study suggest that the A1650G polymorphism in the regulatory region of the CypA gene may be associated with protection from HIV-1 infection, while the 1604G allele may have a weak association with the clinical course of infection in DU.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex.

We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function. Sgt1p physically associates with Skp1p in vivo and in vitro. SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content. Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function. Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p. Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase. sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro. Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.