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PubMed1981

Abundant androgen regulated mRNAs in mouse submandibular gland: cell-free translation of renin precursor mRNA.

Submandibular glands of male mice contain at least four abundant mRNAs that occur at low concentrations in glands of females. The male-specific mRNAs code for polypeptides of 48,000, 43,000, 29,000, and 27,000 MW. Androgenic regulation of these mRNAs is illustrated by their apparent absence in glands of castrate males and by their accumulation in glands of females treated with testosterone. Selective hybrid-arrested translation experiments also indicate reduced levels of these male-specific sequences in female gland cytoplasm. The 48,000 MW male-specific polypeptide is reduced in translation products directed by gland mRNA from C57BL10/J mice (variants deficient in salivary renin), suggesting the corresponding mRNA codes for a renin precursor. The identity of this polypeptide is confirmed by immune selection with renin-specific antibody.

Animals↗
PubMed1998

Novel mutations in the promoter and coding region of the human 5-HT1A receptor gene and association analysis in schizophrenia.

Dysfunction of serotonin systems has been implicated in schizophrenia. In the present study, the human 5-HT1A receptor gene containing the 5' untranslated region was screened in order to detect genetic variations, through which alteration of protein function or level of expression might contribute to schizophrenia. Genomic DNAs were isolated from whole-blood samples of 61 unrelated schizophrenic patients and 100 healthy controls. Genetic variations were screened systematically by single-strand conformational polymorphism (SSCP) analysis, followed by direct sequencing of polymerase chain reaction (PCR) product as well as restriction fragment-length polymorphism (RFLP). The novel mutations (-51T --> C, -152C --> G, -321G --> C, -480delA, and -581C --> A) were found in the 5' untranslated region. Furthermore, we found a novel missense mutation (Gly272Asp) in the coding region in addition to the mutations (Pro16Leu, 294G --> A, and 549C --> T) reported previously. No significant differences in genotype frequencies as well as allele frequencies were found between patients and controls. Our data provided no evidence of association between schizophrenia and the variants in the 5' untranslated region as well as the coding region of the human 5-HT1A receptor gene.

Genetic Markers↗
PubMed1995

GM2 gangliosidosis B1 variant: biochemical and molecular characterization of hexosaminidase A.

The biochemical properties of hexosaminidase A (HexA) and the coding sequence of the alpha-subunit were examined in a patient of Syrian ancestry with the B1 form of Tay-Sachs disease (TSD). The biochemical characteristics of the variant HexA suggest that both active sites are affected by the mutation(s). Kinetic studies with the beta-subunit specific substrate, 4-methylumbelliferyl-beta-D-N-acetylglucosamine (MUG), revealed a significant difference between the Km values. of normal and variant HexA, while no difference was found when the sulfated substrate MUG-6-sulfate (MUGS), which is specific for the alpha-subunit active site, was used. The Vmax values for both substrates were significantly lower in extracts from B1 variant cells than in control extracts, implying a reduced enzyme level in the variant cells. A noncompetitive inhibitor of the reaction with MUGS, N-acetylglucosamine (NAG), induced a significant inhibition (30%) in the mutant cells only. When MUG was used as substrate, variant HexA was found to be more heat stable (T50 = 170 min) than normal HexA (T50 = 65 min). Furthermore, the mutant cell preparation differed from control in the relation between Hex thermosensitivity and protein concentration in the reaction. Two new mutations were identified in exon 5 of the HexA gene: a C496 to G transversion, which produced an Arg166 -->Gly alteration and a deletion of C498 which generated a shift in the reading frame. The patient was a heterozygote for both mutations even though her parents are first cousins. There is no evidence as yet which of these mutations accounts for the B1 phenotype.

Base Sequence↗
PubMed2005

Co-inoculation with two non-infectious cDNA copies of potato spindle tuber viroid (PSTVd) leads to the appearance of novel fully infectious variants.

Potato spindle tuber viroid (PSTVd) is one of the smallest (about 360 nt) infectious plant agents. It is composed of a single-stranded circular non-coding RNA molecule. In the course of previous passage experiments with two intermediate PSTVd variants I2 and I4, three non-infectious clones (I2-50, I4-37 and I4 VI-17) were found. When inoculated separately as cDNAs on tomato "Rutgers" test plants these variants did not induce any visible disease symptoms and did not produce progeny. The presence of such non-infectious variants raises several questions about their origin and biology and to answer them, mixed co-infections with cDNA copies of two non-infectious variants (I2-50, I4-37) were performed. PSTVd infection was observed in seven out of 30 inoculated plants. The progeny isolated from three separate plants contained novel variants, together with the parental I2 and I4 sequences. It is conceivable that the appearance of repaired PSTVd molecules, clearly capable of cell-to-cell movement leading to the systemic infection, results from recombination events. An analysis of the recombinant molecules and comparison with databases identified the specific sites responsible for the restricted infectivity of the I2-50 and I4-37 PSTVd variants. In parallel experiments in which (+) strand PSTVd infectious transcripts were used, no recombinants were observed, and the original I2-50 and I4-37 non-infectious sequences were not detected in the progeny.

DNA, Complementary↗
PubMed1988

The production of normal and variant human glucose-6-phosphate dehydrogenase in cos cells.

Full-length cDNA coding for human glucose-6-phosphate dehydrogenase (G6PD) was inserted into a eukaryotic expression vector containing the immediate early promoter of cytomegalovirus. When this plasmid was introduced into cos cells by transfection it led to the production of high levels of human G6PD. cDNAs containing mutations found in G6PD-deficient individuals were constructed by in vitro mutagenesis and expressed in the same system. Characterization of the G6PD proteins obtained in this way confirmed the primary structure inferred for the variant enzymes. An enzyme in which lysine-205 had been mutated to threonine was produced and found to have no G6PD activity, proving that this lysine residue is essential for enzyme activity in human G6PD.

Cell Line↗
PubMed1996

Gene homology between orf virus and smallpox variola virus.

About 47% identity was observed between the deduced amino acid sequences of a protein encoded by a gene of the parapoxvirus orf virus (OV) strain NZ2 and a 6 kDa protein of unknown function reported to be produced by an open reading frame expressed early after infection by the orthopoxvirus Western Reserve vaccinia virus (VAC); the open reading frame is absent from VAC strain Copenhagen. Examination of sequences reported for variola virus (VAR) strains Bangladesh, India, Congo- 1970, Somalia- 1977 and Garcia- 1966 revealed each encoded a correlate 58 amino acid protein. The open reading frame was not reported in the original analyses of these sequences because a lower limit of 60 amino acids was used to identify potential encoded proteins. Inspection of partial reading frames reported for cowpox virus (CWV) and ectromelia virus (EMV) suggested that these viruses might also code for a correlate of the VAC WR protein. DNA sequencing of cloned fragments of CWV and EMV confirmed that both these orthopoxviruses encode closely related, full length variants of the VAC and VAR open reading frames. The OV homologue is coded in the OV strain NZ2 BamHI-E fragment E2L open reading frame, which we reported is transcribed early postinfection; moreover, analysis of an NZ2 variant showed E2L was absent, indicating that E2L, like the VAC cognate, is nonessential for virus replication in cell culture. The parapoxvirus and orthopoxvirus correlates have about 20% amino acid sequence resemblance to African swine fever virus DNA binding protein p10, suggesting an ancestral relation of genes.

Amino Acid Sequence↗
PubMed2001

Potential role of pharmacogenomics in reducing adverse drug reactions: a systematic review.

CONTEXT: Adverse drug reactions are a significant cause of morbidity and mortality. Although many adverse drug reactions are considered nonpreventable, recent developments suggest these reactions may be avoided through individualization of drug therapies based on genetic information, an application known as pharmacogenomics. OBJECTIVE: To evaluate the potential role of pharmacogenomics in reducing the incidence of adverse drug reactions. DATA SOURCES: MEDLINE English-language only searches for adverse drug reaction studies published between January 1995 and June 2000 and review articles of variant alleles of drug-metabolizing enzymes published between January 1997 and August 2000. We also used online resources, texts, and expert opinion. STUDY SELECTION: Detailed inclusion criteria were used to select studies. We included 18 of 333 adverse drug reaction studies and 22 of 61 variant allele review articles. DATA EXTRACTION: All the investigators reviewed and coded articles using standardized abstracting forms. DATA SYNTHESIS: We identified 27 drugs frequently cited in adverse drug reaction studies. Among these drugs, 59% are metabolized by at least 1 enzyme with a variant allele known to cause poor metabolism. Conversely, only 7% to 22% of randomly selected drugs are known to be metabolized by enzymes with this genetic variability (range, P =.006-P<.001). CONCLUSIONS: Our results suggest that drug therapy based on individuals' genetic makeups may result in a clinically important reduction in adverse outcomes. Our findings serve as a foundation for further research on how pharmacogenomics can reduce the incidence of adverse reactions and on the resulting clinical, societal, and economic implications.

Anticoagulants↗
PubMed1999

Amplification and cloning of infectious bursal disease virus genomic RNA segments by long and accurate PCR.

Improved methods of reverse transcription, polymerase chain reaction (PCR) amplification, and cloning of full-length coding region of both strands of infectious bursal disease virus (IBDV) variant strain E genome were developed. Denaturation of IBDV RNA by heat in the presence of primers and use of a reverse transcriptase lacking RNase-H activity produced full-length coding region and partial non-coding region cDNA copies of the viral genomic segments. Digestion of the RNA component of RNA-cDNA hybrids by RNase-H followed by long and accurate PCR (LA-PCR) amplification of IBDV cDNA in a single step resulted in the synthesis of 3182 base-pairs (bp) of segment A and 2777 bp of segment B cDNA copies of IBDV genome. The resulting amplicons were successfully cloned and sequenced revealing their identity of IBDV. The LA-PCR method can be utilized for the amplification and cloning of the other IBDV strains or isolates and will greatly enhance the availability of sequence information or infectious cDNA copies of IBDV.

Animals↗
PubMed1994

Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin variants, including two PI*Q0 alleles and one deficient PI*M allele.

We have investigated the molecular basis of 15 new alpha 1-antitrypsin (alpha 1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect alpha 1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promoter region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of alpha 1AT:the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3' frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr68(ACC)-->Ile(ATC); and an in-frame trinucleotide deletion delta Phe51 (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal alpha 1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.

Alleles↗
PubMedDate not supplied

T-DNA and transposon tagging in aspen.

We have investigated the somatic activity of the maize Activator (Ac) element in haploid and diploid aspen with the objective of developing an efficient transposon-based system for gene isolation in the model tree species Populus. It was shown that Ac is reinserted, frequently into or near coding regions in aspen, and therefore can be used for gene tagging studies. A number of phenotypic variants were also found following transformation of constructs harbouring the rolC gene. Comparative analyses of T-DNA flanking regions of variants and wild type lines indicate that T-DNA insertion has occurred in or near coding regions. However, the frequency of T-DNA insertion into genes is about one half of the frequency of Ac insertion hitting coding sequences. The results obtained give a proof-of-concept for transposon tagging in a tree system. Given the long generation cycles in tree species, gene tagging strategies are practical only to obtain dominant gain-of-function mutants that do not require selfing or test crossing. In order to obtain recessive loss-of-function mutants, we have regenerated haploid lines from immature pollen. These lines were successfully transformed with a construct containing the rolC transgene from Agrobacterium rhizogenes and Ac element from maize. The results indicate that Ac is also active in haploid aspen and hence can be used in general for gene tagging in trees.

DNA Transposable Elements↗
PubMed1998

Identification and molecular characterization of the new alpha-1-antitrypsin deficient allele PI Y barcelona (Asp256-->Val and Pro391-->His). Mutations in brief no. 174. Online.

To characterize the molecular basis of the "new" alpha1-antitrypsin (alpha1AT) deficient variant, PI Y barcelona, DNA sequence analysis of the coding exons of the alpha1AT gene was carried out using an amplification DNA technique and direct sequencing. The PI Y barcelona allele differs from the normal M1(Val213) allele sequence by two point substitutions: a transversion of GAT TO GTT in exon III in the codon for residue 256, resulting in the amino acid change of Asp256 to Val256, and a transversion of CCC to CAC in exon V in the codon for residue 391, resulting in the amino acid substitution of Pro391 to His391. On isoelectric focusing analysis these substitutions result in a cathodal migration of the "new" variant close to the PI Z. The index case, diagnosed with severe obstructive pulmonary disease, initially phenotyped a PI ZZ, was homozygous for PI Y barcelona. The patient's serum alpha1AT level was 16 mg/dL (normal values 115-220 mg/dL). Inheritance of the PI Y barcelona was confirmed by family study. Amino acid substitution in postion 391 occurs in the C-terminal peptide region, which shows a high degree of homology with the family of serpins. Pro391 is considered to have special relevance in the secretion of alpha1AT.

Alleles↗
PubMed2005

Effects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes.

The human IL-4 receptor alpha chain gene (IL4R) is highly polymorphic and controversial reports have been published with respect to the association of different single nucleotide polymorphisms (SNPs) with atopy markers. Here we analyzed the functional and associational relevance of common IL4R coding SNPs. Transfection of B cell lines expressing the IL-4R variant V75+R576 did not result in enhanced IL-4 induced CD23 expression compared to cell lines expressing the wild type IL-4R alpha chain. Transfection of the IL-4R variant P503 into a murine T cell line did not influence IL-4 induced T-cell proliferation compared to wild type constructs. Analysis of six IL4R coding SNPs (I75V, E400A, C431R, S436L, S503P, Q576R) and common haplotypes (frequency >/=0.05%) in blood donors (n=300) did not indicate a significant association with elevated serum IgE level. Moreover, the most informative IL4R coding SNPs (I75V, C431R, Q576R) and related two- and three-point haplotypes (frequency >/=0.05%) were analyzed in a second, extended group of blood donors (n=689). Again, no significant association with elevated serum IgE was detectable. We conclude that common coding SNPs in the IL4R gene are unlikely to contribute significantly to increased IgE levels and variations outside the coding region may influence atopy susceptibility.

Amino Acid Substitution↗
PubMed2006

Identification of Hepta- and Octo-Uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of SARS-CoV ORF 3a variants.

Programmed frameshifting is one of the translational recoding mechanisms that read the genetic code in alternative ways. This process is generally programmed by signals at defined locations in a specific mRNA. In this study, we report the identification of hepta- and octo-uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of severe acute respiratory syndrome coronavirus (SARS-CoV) ORF 3a variants. SARS-CoV ORF 3a encodes a minor structural protein of 274 amino acids. Over the course of cloning and expression of the gene, a mixed population of clones with six, seven, eight and nine T stretches located 14 nt downstream of the initiation codon was found. In vitro and in vivo expression of clones with six, seven and eight Ts, respectively, showed the detection of the full-length 3a protein. Mutagenesis studies led to the identification of the hepta- and octo-uridine stretches as slippery sequences for efficient frameshifting. Interestingly, no stimulatory elements were found in the sequences upstream or downstream of the slippage site. When the hepta- and octo-uridine stretches were used to replace the original slippery sequence of the SARS-CoV ORF 1a and 1b, efficient frameshift events were observed. Furthermore, the efficiencies of frameshifting mediated by the hepta- and octo-uridine stretches were not affected by mutations introduced into a downstream stem-loop structure that totally abolish the frameshift event mediated by the original slippery sequence of ORF 1a and 1b. Taken together, this study identifies the hepta- and octo-uridine stretches that function as sole elements for efficient +1 and -1 ribosomal frameshift events.

Animals↗
PubMed1997

De novo mutation (Arg98-->Cys) of the myelin P0 gene and uncompaction of the major dense line of the myelin sheath in a severe variant of Charcot-Marie-Tooth disease type 1B.

A point mutation (Arg98-->Cys) of exon 3 coding for the extracellular domain of the myelin protein zero (P0) gene was found in a sporadic case of an eighteen year old Japanese man with a severe variant of Charcot-Marie-Tooth disease type 1B (CMT1B). A de novo mutation was established by parentage testing and analyses of the P0 gene in the family. This patient showed delayed motor development, nonprogressive limb weakness and kyphoscoliosis. In addition to the nerve biopsy findings typical of CMT1B, such as segmental demyelination, marked decrease in the density of myelinated fibers, and frequent onion-bulb formation, ultrastructural examination disclosed uncompaction of the major dense lines with slight widening of the intraperiod distance in the inner layers of the myelin sheath. Although mutations in the extracellular domain of P0 should affect homophilic adhesion between external surfaces of Schwann cell processes, resulting in the separation at the intraperiod lines, our study shows uncompacted major dense lines as a main myelin abnormality where the cytoplasmic domain of P0 resides.

Adolescent↗
PubMed1998

Use of long RT-PCR to characterize splice variant mRNAs.

Recent advances in long reverse transcription (RT)-PCR technology allow the copying of full-length coding regions of large mRNAs in one step. Using long RT-PCR, one can be certain that a given cDNA is derived from a single mRNA. In what to our knowledge is a novel application, we can isolate and characterize splice variants for any given mRNA in a systematic manner. We optimized long RT-PCR to copy the full-length coding region of human multidrug resistance (MDR1) mRNA or the major vault protein (MVP) mRNA in one step, so that only one full-length PCR product was synthesized in each case. Such stringent conditions are necessary to ensure that smaller than full-length products derived from total cell RNA are true splice variants. Twenty MDR1 double-stranded (ds) cDNAs, isolated from either the full-length or one prominent splice-variant DNA band, visualized on agarose gels, were cloned and sequenced. Two were full-length, wild-type in sequence as expected, and the rest were splice-variant mRNAs. Fourteen of the clones were identical and encoded a prominent splice-variant mRNA that can be detected in two tumor cell lines. This approach is shown to be generally applicable to the systematic analysis of splice-variant mRNAs derived from any gene.

Alternative Splicing↗
PubMed1998

Identification of new sequence variants in the leptin gene.

The leptin gene (LEP) has been linked to extreme obesity. However, no common obesity-related gene variants have been found to exist in the LEP. The present study was designed to investigate the LEP for variants by screening both the putative promoter and the coding region of this gene in obese Finnish subjects (n = 200; body mass index, > 27 kg/m2). PCR-amplified DNA samples were subjected to single strand conformation analysis. A G144A substitution in codon 48 and a G328A substitution in codon 110 were identified in two obese subjects, both of whom had very low serum leptin levels. A rare silent C538T polymorphism was detected 33 bp downstream of the translation stop codon (TGA). A common polymorphism A19G was identified in the untranslated exon 1. This polymorphism was not associated with traits of obesity; in agreement, the allele frequencies were similar between 64 normal weight and 141 obese Finns. In summary, this study failed to find a common gene variant in the LEP associated with obesity, but introduces 2 rare mutations associated with very low serum leptin concentrations in 2 obese subjects.

Adult↗
PubMed1997

[Molecular characterization of two variants of alpha-1-antitrypsin deficiency: PI Mpalermo and PI Plovel].

BACKGROUND: Alpha 1 antitrypsin (AAT) is a highly polymorphic protein, having more than 75 different variants. In this work two rare AAT deficient variants were characterized by DNA study. PATIENTS AND METHODS: Members of three generations of two separate families were studied. In family 1, the index case was affected with pulmonary emphysema and presented AAT deficiency (23 mg/dl). In family 2, the index case had a normal pulmonary function, an AAT serum level of 72 mg/dl and a phenotype heterozygous for an AAT variant migrating in the P variant region. The AAT variants were characterized by polymerase chain reaction amplification of the coding exons and direct sequencing of the amplification products. RESULTS: Direct DNA sequencing from a member of family 1 demonstrates that in the exon II of the normal M1 (Val213) allele there was a 3-bp deletion (TTC), corresponding to Phe51 or Phe52. This mutation is characteristic of the Pl Mpalermo variant. In our study, Pl Mpalermo was detected in six members of three generations of this same family. Sequencing of exon III in a member of family 2, identified in the common M1 (Val213) allele a single base substitution of GAT-GTT, with the resulting amino acid change Asp256 for Val256. This mutation characterizes the Pl Plovel allele. The Pl Plovel was also detected in nine members of five others independent families. All of them have AAT serum levels between 80 and 102 mg/dl. None of the studied subjects had clinical evidence of lung disease. CONCLUSIONS: The results of our study show the presence of the two AAT deficient variants in Spain and suggest that the Pl Plovel variant might be more common than expected.

Adult↗
PubMedDate not supplied

Characterization of variant alleles of cytochrome CYP2D6 in a Spanish population.

The CYP2D6 gene codes for a P450 monooxygenase which is involved in the biotransformation of a large number of commonly prescribed drugs. Adverse drug effects and therapeutic failure can be related to abnormal CYP2D6 activity. We investigated the allele and genotype frequencies of cytochrome P4502D6 in a Spanish population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes in our population and to design a feasible CYP2D6 genotyping protocol. The study included 105 healthy unrelated Spanish Caucasian volunteers. CYP2D6 genotyping was performed by a combination of long-PCR, direct sequencing and allele-specific real-time PCR. The frequency of the wild-type CYP2D6*1 allele was 31%. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity showed frequencies of 40.47, 2.38 and 1.90%, respectively. Frequencies of defective alleles *3, *4, *5 and *6 were 0.95, 13.8, 3.33 and 0.95%, respectively. The defective CYP2D6 alleles *7, *8, *12, *14, *15 and *21 were not found. Duplicated CYP2D6 alleles were detected at a frequency of 4.27%. Our protocol allows the identification of the four inactive CYP2D6 alleles (*3, *4, *5 and *6) and the detection of alleles with CYP2D6 *1, CYP2D6 *2 and CYP2D6*4 gene duplications. Testing for this reduced CYP2D6 allele set would facilitate its use in clinical practice by assisting in the development of individualized pharmacotherapy.

Alleles↗