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PubMed1987

cDNA structures of class I human liver alcohol dehydrogenases.

We have determined cDNA structures coding for class I subunits of human liver alcohol dehydrogenase. Two clones have been identified which contain the cDNA sequence coding for the alpha subunit. One of the clones had a 139-nucleotide internal deletion of interest in relation to intron/exon splice junctions, domain borders and evolutionary connections with other dehydrogenases. Different size classes of cDNA clones coding for the beta subunit were characterized with 3' non-coding regions of 213, 590 and 1331 nucleotides. In addition, two unused polyadenylation signals were found, indicating that signals other than AATAAA, are required for 3' end formation. Determination of cDNA structures corresponding to the gamma 1 and gamma 2 subunits makes it possible to explain the kinetic differences between the two allelic subunits in terms of two amino acid replacements. In total, 35 of 374 amino acid residues differ between the class I subunits. Only in the beta pleated sheet region of the coenzyme-binding domain is an almost complete lack of substitutions noted, illustrating the importance of this region. The class II, with pi subunits, has also been determined and shows a much lower extent of homology.

Alcohol Dehydrogenase↗
PubMed1996

Molecular cloning of complex chromosomal translocation t(8;14;12)(q24.1;q32.3;q24.1) in a Burkitt lymphoma cell line defines a new gene (BCL7A) with homology to caldesmon.

Chromosome 12q24.1 is a recurrent breakpoint in high-grade B-cell non-Hodgkin lymphoma (B-NHL). To identify the genes involved at 12q24.1, molecular cloning of a three-way translocation t(8;14;12)(q24.1;q32.3;q24.1) in a Burkitt lymphoma cell line (Wien 133) was performed; all four translocation breakpoints were cloned and sequenced. Analysis of clones encompassing the der(12)(12;14)(q24.1;q32.3) breakpoint showed a CpG island from chromosome 12q24.1 juxtaposed in a tail-to-tail configuration with a productively rearranged Ig VH4-DH-JH5 gene. A total of 4.5 kb of genomic DNA including the CpG island was sequenced and analyzed using gene-identification programs; all three programs identified a potential 92-bp exon within the centromeric boundary of the CpG island. Using this as a probe, an RNA transcript of 3.8 kb, expressed at low levels in a wide variety of normal tissues, was detected. Overlapping cDNA clones were isolated and sequenced. The longest open-reading frame predicted a serine-rich protein of 231 amino acids. This protein, termed BCL7A, exhibited no recognizable protein motifs but showed homology with the actin-binding protein, caldesmon. In Wien 133, the BCL7A breakpoint occurred within the first intron and resulted in a MYC-BCL7A fusion transcript, with exon I of BCL7A being replaced by MYC exon I. The normal, untranslocated allele of BCL7A was also expressed without mutation. One of the 11 other B-NHL cell lines examined with 12q24.1 cytogenetic abnormalities, a mediastinal B-NHL cell line (Karpas 1106), showed biallelic rearrangement within the first intron of BCL7A, which was adjacent to the breakpoint observed in Wien 133. Disruption of the amino-terminus of BCL7A defines a new mechanism in the pathogenesis of a subset of high-grade B-NHL.

Amino Acid Sequence↗
PubMed1985

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.

Adenosine Triphosphatases↗
PubMed1991

Modulation of gene activity by consecutive gene targeting of one creatine kinase M allele in mouse embryonic stem cells.

The cytosolic creatine kinases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue- and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunits, starting at the muscle creatine kinase (CKM) gene which is transcriptionally inactive during early embryogenesis. Using repeated rounds of gene targeting in mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycin resistance (neor) gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several independent doubly targeted clones were isolated which all had insertions in the previously neo-disrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted in gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels in ES cells, led to the formation of the BM isoform of CK in these clones.

Alleles↗
PubMed2005

Human apoE4-targeted replacement mice display synaptic deficits in the absence of neuropathology.

The human APOE*4 allele is associated with an early age of onset and increased risk of Alzheimer's disease (AD). Long before the onset of AD, cognitive deficits can be identified in APOE*4 carriers. We examined neurons in the lateral amygdala of young apolipoprotein (apo) E3 and apoE4 targeted replacement (TR) mice for changes in synaptic integrity. ApoE4 mice displayed significantly reduced excitatory synaptic transmission and dendritic arborization. Despite these changes there were no signs of gliosis, amyloid deposition or neurofibrillary tangles in these mice. To our knowledge, this is the first study to suggest that cognitive deficits in APOE*4 carriers are due to inherent defects in synaptic function that appear prior to any age-dependent markers of neuropathology.

Affective Symptoms↗
PubMed2004

Genetic makeup of the DR region in rhesus macaques: gene content, transcripts, and pseudogenes.

In the human population, five major HLA-DRB haplotypes have been identified, whereas the situation in rhesus macaques (Macaca mulatta) is radically different. At least 30 Mamu-DRB region configurations, displaying polymorphism with regard to number and combination of DRB loci present per haplotype, have been characterized. Until now, Mamu-DRB region genes have been studied mainly by genomic sequencing of polymorphic exon 2 segments. However, relatively little is known about the expression status of these genes. To understand which exon 2 segments may represent functional genes, full-length cDNA analyses of -DRA and -DRB were initiated. In the course of the study, 11 cDRA alleles were identified, representing four distinct gene products. Amino acid replacements are confined to the leader peptide and cytoplasmatic tail, whereas residues of the alpha1 domain involved in peptide binding, are conserved between humans, chimpanzees, and rhesus macaques. Furthermore, from the 11 Mamu-DRB region configurations present in this panel, 28 cDRB alleles were isolated, constituting 12 distinct cDRA/cDRB configurations. Evidence is presented that a single configuration expresses maximally up to three -DRB genes. For some exon 2 DRB sequences, the corresponding transcripts could not be detected, rendering such alleles as probable pseudogenes. The full-length cDRA and cDRB sequences are necessary to construct Mhc class II tetramers, as well as transfectant cell lines. As the rhesus macaque is an important animal model in AIDS vaccine studies, the information provided in this communication is essential to define restriction elements and to monitor immune responses in SIV/simian human immunodeficiency virus-infected rhesus macaques.

Alleles↗
PubMed1995

Homologous gene replacement in Physarum.

The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD delta 1, of the ardD actin gene; ardD delta 1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD+ with ardD delta 1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD delta 1 in addition to ardD+, but in two (5%), ardD+ was replaced by a single copy of ardD delta 1. This is the first example of homologous gene replacement in Physarum. ardD delta 1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD delta 1 on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene.

Actins↗
PubMed1997

Comprehensive method for the typing of HLA-A, B, and C alleles by direct sequencing of PCR products obtained from genomic DNA.

Molecular testing is gradually replacing standard typing techniques in the field of HLA because it allows higher resolution, which has significant functional implications. Although several techniques have been so far described for this purpose, the definitive means to determine which alleles are present in a particular sample is to identify their sequence. We describe a simplified method for typing HLA-A, B, and C alleles by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA that could allow large-scale handling of samples for clinical use. The template is the product of a nested PCR. A first round of PCR amplifications from genomic DNA is performed with three different sets of primers, one pair specific for each locus. The PCR products encompass exons 2 and 3, the regions of interest to determine the allele present. These fragments are a mixture of both alleles present in one locus. In a second round of PCRs using the first fragment as template, exons 2 and 3 are separately amplified and simultaneously tailed with sequences corresponding to fluorescent-labeled commercial primers. The sense and antisense sequence of each exon is obtained and compared with a database of all known HLA-A, B, or C alleles. Heterozygous positions are determined and the most probable alleles assigned. This simplified procedure has the practical advantage of allowing high-resolution typing of clinical material by utilizing the same genomic DNA used for standard molecular typing of HLA class I.

Alleles↗
PubMed2006

Genomic diversity and evolution within the species Streptococcus agalactiae.

Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.

Adult↗
PubMed1999

A cross-ethnic analysis of risk factors for AD in white Hispanics and white non-Hispanics.

BACKGROUND: The prevalence of AD appears to vary widely in different ethnic groups. Certain risk factors for AD are well established for the general population, but there is little information regarding the relevance of these risk factors in specific ethnic groups. OBJECTIVE: The authors examined the risk of AD associated with the APOE-epsilon4 allele, the APOE-epsilon2 allele, smoking, alcohol consumption, history of hypertension, low educational level, estrogen replacement therapy, and history of head trauma with loss of consciousness among samples of white non-Hispanics (WNH) (392 AD patients, 202 normal subjects) and white Hispanics (WHIS) (188 AD patients, 84 normal controls). DESIGN: This was a case-control study of patients evaluated at an outpatient memory disorders clinic and control subjects recruited from a free memory screening offered to the community. RESULTS: Increased risk for AD was associated with the APOE-epsilon4 allele after controlling for age, education, and gender among WNH (OR = 3.5; 95% CI = 2.3 to 5.5) and WHIS (OR = 3.1; 95% CI = 1.7 to 5.8). No protective effect was conferred by the APOE-epsilon2 allele, although this relationship approached significance among WNH (p = 0.02). Low levels of education increased the risk for AD among WNH (OR = 3.1; 95% CI = 1.8 to 5.9) but not WHIS. Alcohol use and hypertension approached significance as risk factors in WNH (p < 0.05) but not WHIS. Estrogen replacement treatment approached significance as a protective factor in both ethnic groups (p < 0.05). CONCLUSIONS: Although the APOE-epsilon4 allele is a risk factor for AD among WHIS and WNH, other risk factors such as low education and hypertension appear to be important only for WNH. Risk factors for AD reported or suggested previously that were not confirmed by this study include smoking and head trauma with loss of consciousness.

Aged↗
PubMed1988

Recent origin for a thermostable alcohol dehydrogenase allele of Drosophila melanogaster.

The nucleotide sequence of the Fast-Chateau Douglas isolate of the thermostable alcohol dehydrogenase allele is compared with the sequences of the Slow and Fast alleles of Drosophila melanogaster. Conceptual translation of the FChD sequence indicates that the thermostable polypeptide has the diagnostic FAST amino acid replacement at residue 192 and an additional replacement of serine for proline at residue 214. This suggests a Fast origin for the thermostable Adh allele. However, some of the biochemical properties of the FCHD protein resemble those of the SLOW rather than the FAST polypeptides. The serine for proline replacement confers upon the thermostable polypeptide substrate specificities and some kinetic parameters similar to the SLOW protein. The same replacement substitution within the third coding exon also appears to alter the ADH protein concentration to a level similar to the SLOW polypeptide and the probable effect is at the level of mRNA concentration. The low level of nucleotide sequence variation, other than that leading to the amino acid substitution, suggests a recent origin for the thermostable allele. The time since divergence of the FChD sequence from Fast is estimated to be approximately 260,000-470,000 years.

Alcohol Dehydrogenase↗
PubMed1999

Characterization of a double gene disruption in the LYS2 locus of the pathogenic yeast, Candida albicans.

The 4.2 kbp LYS2 gene of Candida albicans encodes the 150 kDa subunit of the 180 kDa heterodimeric enzyme alpha-aminoadipate reductase. To facilitate structural and functional studies of the LYS2 gene, in this investigation both alleles were sequentially disrupted in C. albicans CAI4. The disruptions were performed using a targeting vector that contained a 2.2 kbp portion of LYS2 from which a 600-bp fragment had been deleted and replaced with a hisG-URA3-hisG cassette. Disruption of both alleles of the LYS2 locus was confirmed by Southern hybridization with appropriate probes. To determine the physiological effect of the LYS2 disruption, cell extracts were assayed for the LYS2-encoded alpha-aminoadipate reductase activity. The parent, heterozygote and LYS2-disrupted strains exhibited significant, partial and no activity, respectively. Transformation of the LYS2-disrupted strain with the LYS2 gene reversed the mutant phenotype as shown by growth on selective media and restored alpha-aminoadipate reductase activity. These results demonstrated that the LYS2 gene of C. albicans is responsible for the synthesis of functional alpha-aminoadipate reductase.

Aldehyde Oxidoreductases↗
PubMed2000

Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions.

The activins (dimers of betaA or betaB subunits, encoded by the genes Inhba and Inhbb, respectively) are TGF-beta superfamily members that have roles in reproduction and development. Whereas mice homozygous for the Inhba-null allele demonstrate disruption of whisker, palate and tooth development, leading to neonatal lethality, homozygous Inhbb-null mice are viable, fertile and have eye defects. To determine if these phenotypes were due to spatiotemporal expression differences of the ligands or disruption of specific ligand-receptor interactions, we replaced the region of Inhba encoding the mature protein with Inhbb, creating the allele Inhbatm2Zuk (hereafter designated InhbaBK). Although the craniofacial phenotypes of the Inhba-null mutation were rescued by the InhbaBK allele, somatic, testicular, genital and hair growth were grossly affected and influenced by the dosage and bioactivity of the allele. Thus, functional compensation within the TGF-beta superfamily can occur if the replacement gene is expressed appropriately. The novel phenotypes in these mice further illustrate the usefulness of insertion strategies for defining protein function.

Activins↗
PubMed1985

N-ras gene activation in the RD human rhabdomyosarcoma cell line.

A transforming N-ras allele was molecularly cloned from the RD human rhabdomyosarcoma cell line, and the nature of its activation studied. Construction of chimeric recombinants between the RD-transforming allele and a normal human allele enabled us to localize the alteration responsible for the activation to the second exon of the N-ras gene. The nucleotide sequence of this exon, when compared to that of the normal allele, revealed a single difference at the 61st amino acid position of the encoded protein; the CAA codon for glutamine in the normal allele was mutated to a CAT codon for histidine in the RD-transforming allele. This result is the first description of a histidine replacing glutamine in the 61st position and provides further evidence that the 61st amino acid is one of the preferential sites for N-ras activation.

Alleles↗
PubMed1992

Amino acid substitutions in the CytR repressor which alter its capacity to regulate gene expression.

In Escherichia coli, transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. Transcription initiation of cistrons in this gene family is activated by cyclic AMP-catabolite activator protein (cAMP-CAP), repressed by the CytR protein, and induced by cytidine. A random proofreading mutagenesis procedure and a genetic screen using udp-lac fusions have allowed the identification of distinct regions of the 341-amino-acid CytR polypeptide that are critical for repression of gene expression and response to induction. Determination of the ability of various CytR mutants to control gene expression in vivo indicated that the intrinsic affinity of the CytR protein for operator DNA is gene specific and that efficient repression of transcription by wild-type CytR is dependent on the interaction of CytR with cAMP-CAP. CytR mutants that were cytidine induction defective (CID) were characterized; these mutant proteins had only Asp-281 replaced. Data obtained with cytR delta M149, a dominant negative allele, indicated that the native CytR repressor is an oligomeric protein. Representative cytR mutations were combined with cytR delta M149, and the resulting hybrid repressors were tested for transdominance in a CytR+ E. coli strain. Amino acid substitutions A209E and C289Y suppressed the transdominance of CytR delta M149, suggesting that these replacements alter the normal protein contacts involved in repressor subunit-subunit association. In contrast, amino acid substitutions located in the N-terminal portion of the CytR protein had no effect on the transdominance of CytR delta M149. The results from this study suggest that the CytR repressor is an oligomeric, allosteric protein in which conformational changes are required for repression and derepression.

Allosteric Regulation↗
PubMed1986

The gamma 1 and gamma 2 subunits of human liver alcohol dehydrogenase. cDNA structures, two amino acid replacements, and compatibility with changes in the enzymatic properties.

cDNA clones corresponding to two alleles of the ADH3 locus were identified by hybridization with synthetic oligodeoxyribonucleotides specific for class I human liver alcohol dehydrogenase. Sequences were determined for a 1457-nucleotide cDNA, covering the whole gamma 2-coding region, and a 1224-nucleotide cDNA, including the region coding for amino acid residues 53-374 of the gamma 1 subunit. Two amino acid replacements between the gamma 1 and gamma 2 subunits were identified. At position 349, isoleucine in gamma 1 instead of valine in gamma 2 is a conservative exchange of a superficial residue which has been ascribed no special importance. The other exchange, at position 271, arginine in gamma 1 and glutamine in gamma 2, explains differences in enzyme properties. Electrophoretically, it is consistent with the less cathodic mobility of the gamma 2 subunit. Functionally, the location of the exchange at the surface of the coenzyme-binding pocket may influence the dissociation of the reduced coenzyme.

Alcohol Dehydrogenase↗
PubMed1994

Targeting of an enhancer trap to vestigial.

The vestigial gene (vg+) is required for normal wing development and is expressed in a spatially distinct pattern in imaginal discs. We have exploited a general property of P element alleles to target an enhancer trap to the 5' region of the gene. By replacing the P element resident at this site in vg21 with a P element carrying a lacZ reporter gene, the vglacZ1 allele was selected on the basis of its increased mutant phenotype. In contrast to vg+ expression, which occurs primarily in the presumptive wing margin and hinge, beta-galactosidase expression in vglacZ1 wing discs is localized to the dorsal wing surface and displays homologous haltere expression. The targeting of P element enhancer traps could be readily extended to other genes with low rates of primary P element insertion.

Animals↗
PubMed2002

A polymorphism in the growth hormone-releasing hormone receptor gene: clinical significance?

Two forms of the growth hormone-releasing hormone (GHRH) receptor (GHRH-R) exist in terms of a polymorphism at codon 57. The most common allele possesses GCG, coding for Ala. This codon can also be ACG, replacing the Ala with Thr. The present study demonstrates that the latter occurs in about 20% of pituitary somatotrophinomas, removed from patients with acromegaly. Somatotrophinomas possessing the alternative allele respond, on average, more strongly to GHRH in terms of GH secretion in vitro than tumors which are homozygous for the more common allele. The distribution of the two allelic forms of the GHRH-R did not significantly differ between acromegalic and non-acromegalic subjects. Thus, while the alternative allelic forms may, at least partially, contribute to the variable response of serum GH levels to i.v. GHRH observed in acromegalic and normal subjects, it is unlikely that subjects possessing the rarer form containing Thr in place of Ala at residue 57 are at increased risk of developing acromegaly.

Acromegaly↗