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PubMed1984

The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli.

Parameters influencing the efficiency of expression of the human immune interferon (IFN-gamma) gene in E. coli were studied by comparing a series of eight in vitro-derived gene variants. These contained all possible combinations of silent mutations in the first three codons of the mature IFN-gamma polypeptide coding sequence. Expression levels varied up to 50-fold among the different constructions. Comparison of messenger RNA secondary structure models for each variant suggested that the presence of stem-loop structures blocking the translation initiation signals could drastically decrease the efficiency of IFN-gamma synthesis. With variants displaying no stable mRNA secondary structure in the region, a C----U transition at position +11 after the AUG resulted in a 5-fold increase in expression indicating that RNA primary structure also plays an important role in expression. In addition we demonstrate that, in this system, a spacing of 8 nucleotides between the Shine-Dalgarno region and AUG was optimal for gene expression and that the steady-state production level of IFN-gamma rose exponentially with increasing rate of synthesis.

Base Sequence↗
PubMed2000

Expression of hGSTP1 alleles in human lung and catalytic activity of the native protein variants towards 1-chloro-2,4-dinitrobenzene, 4-vinylpyridine and (+)-anti benzo[a]pyrene-7,8-diol-9,10-oxide.

The human glutathione S-transferase (GST) P1 alleles coding for Val(105) (hGSTP1*B and/or P1*C) are over- represented in lung cancer patients. However, the corresponding recombinant Val(105) protein variants tend to show higher catalytic activity than the Ile(105) variants towards bay-region diol epoxides that are thought to be etiological agents in lung cancer. We have examined 29 normal human lung samples with respect to several factors that could confound relationships between hGSTP1 allele type and cancer susceptibility, namely, inter-individual and allele-specific variation of hGSTP1 expression, and differences between the catalytic properties of the native and recombinant hGSTP1-1 variant protein products. hGSTP1 expression varied 7-fold among individuals but was independent of hGSTP1*A, P1*B or P1*C allele type. hGST subunits A1, A2, M1 and M3 were minor components, similarly variable in expression. Despite this variability of expression, the levels of hGSTP1 expression linearly correlated with those of the next most highly expressed GST, hGSTM3, even though the genes for these GSTs are on different chromosomes. Differences between the native protein variants, using 1-chloro-2,4-dinitrobenzene and (+)-anti-benzo[a]pyrene diolepoxide as substrates, were more marked than those between the recombinant variants. However, the order of differential catalytic specificity was the same for native and recombinant variants. Neither the expression of the hGSTP1 alleles nor the catalytic properties of the protein variants appears to provide a simple mechanistic rationale for the observed over-representation of the hGSTP1*B and/or 1*C alleles in lung cancer.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2025

Mistranslating tRNA variants impact the proteome and phosphoproteome of Saccharomyces cerevisiae.

Transfer RNAs (tRNAs) ensure accurate decoding of the genetic code. However, mutations in tRNAs can lead to mis-incorporation of an amino acid that differs from the genetic message in a process known as mistranslation. As mistranslating tRNAs modify how the genetic message is decoded, they have potential as therapeutic tools for diseases caused by nonsense and missense mutations. Despite this, they also produce proteome-wide mis-made proteins which can disrupt proteostasis. To better understand the impact of mistranslating tRNA variants, we profile the proteome and phosphoproteome of yeast expressing three different mistranslating tRNAs. While the overall impacts were similar, the extent of growth defects and proteome changes varied with the substitution type. Although the global impacts were modest, mistranslation influenced key cellular processes, including proteostasis, cell cycle and translation. These findings highlight the need to consider cellular consequences when developing mistranslating tRNAs for therapeutic applications.

mass spectrometry↗
PubMed2003

Bcl-rambo beta, a special splicing variant with an insertion of an Alu-like cassette, promotes etoposide- and Taxol-induced cell death.

The exonization of an Alu-like element into a coding sequence is unique to primates and this phenomenon distinguishes our genome from other mammals. Here, we report the presence of a special splicing variant of a proapoptotic protein Bcl-rambo in human lymph node, designated as Bcl-rambo beta. This variant contains a 98 bp Alu-like sequence which acts as an exon. There exists an in-frame stop codon within this inserted Alu-like cassette, resulting in generation of a premature protein of 104 amino acid residues. Unlike the Bcl-rambo, Bcl-rambo beta is lacking of the BH1, BH2 and BH3 motifs and becomes a BH4-only protein. Bcl-rambo beta is detected in several adult human tissues such as heart, lymph node and cervix but is absent in human brain tissue. In addition, Bcl-rambo beta is found not to be associated with mitochondria due to the absence of its C-terminal membrane anchor region. Nevertheless, this cytosol-localized protein is capable of promoting etoposide- and Taxol-induced cell death. Although the exact function of the Alu sequence is not fully characterized, the Alu element within the Bcl-rambo beta appeared to contribute to the proapoptotic capability, since removing of the Alu sequence from Bcl-rambo beta abrogates its ability to induce cell death. Our data support the speculation that the Alu element insertion during the splicing process may play an important role in the generation of protein diversity in primate cells by a yet uncharacterized mechanism.

Alu Elements↗
PubMed2000

Studies on linkage and association of atopy with the chromosomal region 12q13-24.

BACKGROUND: Several studies have shown linkage of bronchial asthma, allergic rhinitis and total serum IgE concentration to the chromosomal region 12q13-24 in ethnical diverse populations. This region harbours a number of candidate genes for asthma and atopy, including stem cell factor (SCF), leukotriene A4 hydrolase (LTA4H), thyroid receptor 2 (TR2), and signal transducer and activator of transcription 6 (STAT6). However, the same region was shown as well to be linked to other diseases with inflammatory character. So far no variants in any of these genes have been published which would allow association studies and confirm the pathogenicity of any of these genes. OBJECTIVE: We wanted to test for linkage of the chromosomal region 12q13-24 with the atopic phenotype without regard to clinical manifestations. Furthermore we screened for common nucleotide polymorphisms in candidate genes to enable association studies. METHODS: We employed sib-pair linkage analysis and transmission disequilibrium testing with regard to four highly polymorphic microsatellite markers in 12q13-24 in atopic nuclear families. In addition, we looked for polymorphisms in the genes coding for SCF, LTA4H, TR2 and STAT6 performing SSCP-analysis and direct genomic sequencing. RESULTS: We found no evidence for linkage of the genomic region 12q13-24 to elevated total serum IgE levels, specific sensitization to common inhalant allergens or atopy. Furthermore we identified three nucleotide polymorphisms including one common variant in the gene coding for SCF. No association of this polymorphism and any of the atopic phenotypes was seen. CONCLUSION: We conclude from our data that genes in the chromosomal region 12q13-24 and in particular SCF are unlikely to exert a major effect on the induction of the atopic phenotype in our Caucasian population. However, we did not focus on the asthmatic and thereby inflammatory aspect of atopy which might explain these results in contradiction to previous studies.

Chromosomes, Human, Pair 12↗
PubMed1994

Mutational events in consecutive passages of hepatitis A virus strain GBM during cell culture adaptation.

In order to study the adaptation of hepatitis A virus (HAV) in cell culture, we examined the mutational events of the genome in early passages of HAV strain GBM propagated either in FRhK-4 cells (fetal rhesus monkey kidney-derived) or in human embryonic kidney (HEK) and human embryonic fibroblast cells (HFS) in relation to their growth characteristics. Sequence analysis of the nucleotide region encoding 2B, 2C, and the beginning of 3A as well as the nucleotide region encompassing the 5' noncoding region (5'NCR) of the genome was performed on consecutive virus passages after amplification of the viral RNA from the cell culture supernatant by antigen-capture PCR. By the 2nd passage of the GBM variants cultured in FRhK-4 or in HEK cells we found a mutation at nucleotide position 3889 (2B coding region) which results in an amino acid substitution from alanine to valine. Further mutations present in the 2B/2C region of the cell culture-adapted GBM variants differ from each other and occur after the 10th or even the 40th virus passage. Another early change, an in frame deletion of nine nucleotides in the 3A region, appeared in the 5th virus passage only in GBM cultured on FRhK-4 cells. This genome region showed different mutations in the virus passages on HEK and HFS cells. The 5'NCR of the cell culture-adapted GBM variants, in contrast, did not show any mutations before the 8th virus passage. The faster and more efficient growth of the HAV strain GBM during successive propagation on cell cultures seems to correlate with the appearance of mutations in the investigated genome regions.

Adaptation, Physiological↗
PubMed1995

Characterization of cDNAs coding for glucose phosphate isomerase and phosphoglycerate kinase in Chinese hamster ovary cell line CHO-K1 and identification of defects in R1.1.7, a glycolysis-deficient variant of CHO-K1.

Full-length cDNAs for glucose phosphate isomerase (GPI) and phosphoglycerate kinase (PGK) of the Chinese hamster ovary cell line CHO-K1 have been characterized using RT-PCR and cycle sequencing of the PCR-amplified templates. Mutations in both genes have been identified in a glycolysis-deficient Chinese hamster ovary cell line, R1.1.7, derived from CHO-K1 cells.

Animals↗
PubMed2002

A candidate gene approach within the susceptibility region PCaP on 1q42.2-43 excludes deleterious mutations of the PCTA-1 gene to be responsible for hereditary prostate cancer.

OBJECTIVE: The Prostate Carcinoma Tumor Antigen-1 (PCTA-1) is located at the prostate cancer susceptibility locus on chromosome 1q42.2-43 (PCaP). In this candidate gene approach, we searched for deleterious mutations within the PCTA-1 gene and its promoter. MATERIALS AND METHODS: Seventy-seven familial prostate cancer cases from 36 German and French pedigrees were screened for germline mutations in the PCTA-1 gene using enzymatic mutation detection (EMD). Putative missense mutations were genotyped by RPLP and ddNTP primer extension assays in 88 controls to assess allele frequencies and haplotypes. RESULTS: Several sequence variants were found but none of the findings indicated a deleterious mutation. Three affected brothers showed an intronic variation, which may interfere with correct splicing. Four non-conservative SNPs were characterized, coding for the amino acid alterations Y19F, C36R, V56M and S184R. All exchanges were found in controls with common allelic frequencies of at least 28%. Haplotype definition including six SNPs within the PCTA-1 gene revealed a complete linkage disequilibrium. Low haplotype diversity leads to a predominance of only two peptide variants of the PCTA-1 protein, coded by 95% of all chromosomes. CONCLUSIONS: PCTA-1 is not a classical high risk gene with deleterious mutations predisposing to hereditary prostate cancer. Its contribution to prostate cancer susceptibility as a low risk factor in sporadic disease has to be assessed in larger samples by association studies.

Galectins↗
PubMed2004

Stability of JC virus coding sequences in a case of progressive multifocal leukoencephalopathy in which the viral control region was rearranged markedly.

CONTEXT: It is generally accepted that JC virus variants in the brains of patients with progressive multifocal leukoencephalopathy are generated from archetypal strains through sequence rearrangement (deletion and duplication, or deletion alone) in the control region. This change is thought to occur during persistence of JC virus in patients. OBJECTIVE: The present study was performed to ascertain whether amino acid substitution in the viral proteins is involved in the generation and propagation of JCV variants with rearranged control regions. DESIGN: Many complete JC DNA clones were established from brain tissues (cerebellum, occipital lobe, and brainstem) autopsied in a case of progressive multifocal leukoencephalopathy in which multiple distinct control sequences were detected. Control and coding sequences were determined and compared among the JC DNA clones. RESULTS: Twenty-eight control-region and 20 coding sequences of JC virus were compared. Five rearranged control sequences were detected, but they could be classified into 3 groups that shared common structural features. Viral coding sequences were identical among clones with different control regions and among clones derived from different brain regions. CONCLUSION: In the present case, nucleotide substitution in the viral coding regions (and resultant amino acid change in the viral proteins) was involved neither in the genesis of rearranged JC virus variants nor in the proliferation of demyelinated lesions in the brain.

Adolescent↗
PubMed1999

Characterization of single-nucleotide polymorphisms in coding regions of human genes.

A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.

Alleles↗
PubMed1992

Direct selection of hepatoma cell variants deficient in alpha 1-antitrypsin gene expression.

Expression plasmids containing the human alpha 1-antitrypsin (alpha 1 AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT- HPRT- rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes in cis, as only three of 20 clones tested were affected for expression of alpha 1AT mRNA. In contrast, double selection yielded predominantly trans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal alpha 1AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1 trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.

Adenine Phosphoribosyltransferase↗
PubMed1996

Fish glucocorticoid receptor with splicing variants in the DNA binding domain.

Here we describe the isolation of a rainbow trout cDNA containing an entire GR coding region. Although the encoded protein is highly homologous to other GRs, especially in its DNA binding domain, it contains a nine amino acid insertion between the two zinc fingers. This novel form is found in all rainbow trout tissues examined; however, the testis also contains a splice variant lacking this insert, making it completely continuous to other GRs. In transient transfection assays of cultured cells, the two rainbow trout GR variants activated transcription from the glucocorticoid-responsive mouse mammary tumor virus promoter to comparable levels.

Alternative Splicing↗
PubMed1988

Drosophila has a single copy of the gene encoding a highly conserved histone H2A variant of the H2A.F/Z type.

The Tetrahymena histone H2A variant designated hv1 is localized exclusively in the transcriptionally active macronucleus and is absent from the quiescent micronucleus (1). A cDNA clone of the hv1 gene (2) was used to screen a Drosophila cDNA library. A cross-hybridizing clone was recovered and shown by sequence analysis to code for a protein homologous to hv1 as well as to the chicken H2A variant, H2A.F (3), the sea urchin H2A variant, H2A.F/Z (4) and the mammalian H2A variant H2A.Z (5). Southern analysis of Drosophila genomic DNA indicates that the H2AvD (H2A variant Drosophila) gene is present in one copy. In situ hybridization places the locus at 97CD on chromosome 3, while the S-phase regulated histone genes are on chromosome 2 (6). Thus the Drosophila H2A variant should be accessible to genetic analysis, which will enable its function to be determined.

Amino Acid Sequence↗
PubMed2000

Sequence variation within the neuropeptide Y gene and obesity in Mexican Americans.

OBJECTIVE: Recently, we reported evidence for linkage between neuropeptide Y (NPY) and both obesity and several obesity-related quantitative measures in a sample of Mexican Americans from Starr County, Texas. The purpose of this study was to investigate putative variation within the coding and promoter regions of NPY. RESEARCH METHODS AND PROCEDURES: Five young, obese individuals (body mass index [BMI] 33 to 45 kg/m2, age 14 to 30 years); five adult, lean individuals (BMI 20 to 26 kg/m2, age 39 to 65 years); and five sibling pairs sharing no alleles that were identical by descent at a marker locus proximal to NPY were selected for fluorescence-based sequencing of approximately 1100 base pairs (bp) immediately 5' from the start site and all four exons of NPY. We identified a total of eight variant sites, including a 2-bp insertion/deletion (I/D) within a putative negative regulatory region (-880I/D) and a 17-bp deletion at the exon 1/intron 1 junction (69I/D). The -880I/D and 69I/D variants were typed in a separate random sample of Mexican Americans (N = 914) from Starr County, Texas. RESULTS: Analyses of variance resulted in a significant association between -880I/D and waist-to-hip ratio (p = 0.041) in the entire sample and between -880I/D and BMI (p = 0.031), abdominal circumference (p = 0.044), and waist-to-hip ratio (p = 0.041) in a non-obese subsample (BMI < 30 kg/m2, n = 594). The 69I/D variant was observed in only one pedigree and does not appear to segregate with obesity within this pedigree. DISCUSSION: This study reports newly identified common human sequence variation within the regulatory and coding sequence of NPY. Several variants were observed, and of those tested, the -880I/D promoter region variant may influence body fat patterning in non-obese individuals but does not appear to play a major role in the etiology of common forms of obesity in this population.

Adolescent↗
PubMed2000

Identification of a naturally occurring Pro385-Ser385 substitution in the GABA(A) receptor alpha6 subunit gene in alcoholics and healthy volunteers.

In the rat, variation in alcohol and benzodiazepine sensitivity has been correlated with an inherited variant of the GABA(A)alpha6 receptor. Our goal was to identify polymorphisms in the human GABA(A)alpha6 receptor gene and determine whether a variant of the receptor is associated with alcoholism. The GABA(A)alpha6 receptor gene coding region was screened in 80 unrelated patients with alcoholism using single strand conformational polymorphism analysis. For rapid genotyping, a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay was developed. A relatively abundant amino acid substitution and three synonymous DNA substitutions were detected. The synonymous variants, 35A>G, 665A>G, and 1031G>C had rare-allele frequencies of 0.25, 0.02, and 0.47, respectively. The Pro385Ser substitution is located in the second intracellular domain of the receptor adjacent to a putative phosphorylation site. Pro385Ser has rarer allele frequencies of 3.3% and 4.8% in 196 Finnish alcoholic patients and 189 controls, respectively (P = NS). A naturally occurring non-conservative Pro385Ser was detected in the GABA(A)alpha6 receptor. The variant is not associated with alcoholism.

Alcoholism↗
PubMed1998

HLA-C high resolution typing: analysis of exons 2 and 3 by sequence based typing and detection of polymorphisms in exons 1-5 by sequence specific primers.

HLA-C high resolution sequence based typing developed in this study involves a unique DNA amplification encompassing exon 1 to intron 3 and four fluorescent sequencing reactions covering exon 2 and 3. Both dye primer and dye terminator sequencing techniques were performed and results compared. This approach allowed the identification of all of the 50 HLA-C allelic variants so far described, except for two allele pairs that are distinguished by non-coding nucleotide changes (Cw*12021 = 12022, Cw*15051 = 15052) and three allele pairs (Cw*0701 = 706, Cw*1701 = 1702 and Cw*1801 = 1802) that share the same nucleotide sequence in exon 2 and 3. For complete subtyping of these allelic variants, an amplification based on sequence specific primers (PCR-SSP) was used. No ambiguous heterozygous combinations of alleles were detected in our panel so far. HLA-C typing data obtained by this method were compared with data from serological and low resolution PCR-SSP typing, which had been performed previously on the samples sequenced.

Base Sequence↗
PubMed2005

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1.

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.

Agglutinins↗
PubMed1998

De novo mutations and allelic diversity at minisatellite locus D7S22 investigated by allele-specific four-state MVR-PCR analysis.

We have studied the allelic diversity and de novo mutations at the hypervariable minisatellite locus D7S22. A four-state minisatellite variant repeat unit mapping by PCR (MVR-PCR) method was developed for this purpose, and a substitution polymorphism close to the repeat array was used to design allele-specific flanking primers to study individual haplotypes in genomic DNA. A total of 150 alleles from different allele size groups and flanking haplotypes were mapped. On average, MVR-codes extending 65 repeats (2.4 kb) into the repeat array were obtained. The interspersion patterns of variant repeats were highly polymorphic. However, subgroups of alleles close in size and with identical flanking haplotype revealed common MVR-code characteristics indicating a close evolutionary relationship. Unlike the situation in many other hypervariable minisatellites, no polarized variability was revealed at this minisatellite locus. Fifty four small families with D7S22 de novo mutations were analysed by MVR-PCR. The sites where the length change occurred were revealed in 22 cases, while in 32 cases the mutation obviously occurred further into the repeat array. In agreement with a non-polar distribution of the allelic variation, there was no evidence for a hypermutable hot spot for mutation within the repeat array. Comparison of MVR-codes in the mutant and progenitor in gain mutations indicated that at least one, possibly four cases, reflected inter-allelic events. Together with evidence from DNA sequencing of alleles of <2 kb, this indicates that as many as half of the gain mutations might be inter-allelic events in D7S22. Based on these results, different factors which might affect the mutation rate are discussed.

Alleles↗