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Biomedical subjects

M Schweizer

Publications and source records attributed to M Schweizer.

At least 127 records · Page 7Linked to original sources
PubMed1991

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated.

The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 bp encoding a protein of 2076 amino acids and 229,980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS beta-subunit. The sequential order of the five FAS1-encoded enzyme The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3' end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 bp and encodes a protein of 2051 amino acids and 228,667 Da molecular weight.

Amino Acid Sequence↗
PubMed1991

Mechanism of monoclonal antibody inhibition/stimulation of reactions catalyzed by cytochrome P450IIB1.

We describe two monoclonal antibodies (MAbs) against rat cytochrome P450IIB1 and investigate the mechanisms by which they influence P450IIB1-mediated catalysis. MAb ce9 partially inhibits the activities toward p-nitroanisole, 7-ethoxycoumarin, and benzphetamine as well as NADPH oxidation. These findings can be explained by the observation that ce9 cross-links P450 to form large aggregates resulting in the inhibition of the functional interaction with NADPH cytochrome P450 reductase. Binding of ce9 to P450IIB1 does not affect the spin state of the P450 heme, as revealed by comparing the magnetic circular dichroism (MCD) spectra of free and antibody-bound P450IIB1. On the other hand, the second antibody tested, MAb 14E10, induces a remarkable low to high spin transition upon binding to P450IIB1, as shown by MCD difference spectroscopy. This MAb stimulates activities toward p-nitroanisole and 7-ethoxycoumarin without affecting the rate of NADPH oxidation. This observation indicates that MAb 14E10 may increase the efficiency of electron utilization by P450IIB1. Benzphetamine metabolism remains unchanged in the presence of MAb 14E10.

Animals↗
PubMed1991

The intron enhancer of the immunoglobulin kappa gene activates c-myc but does not induce the Burkitt-specific promoter shift.

In Burkitt's lymphoma cells the c-myc gene locus is consistently fused to the constant region of one of the immunoglobulin genes by chromosomal translocation. The translocated c-myc gene is transcriptionally activated and preferentially transcribed from the P1 promoter whenever the exon-intron structure of c-myc remains intact. In order to define elements involved in this promoter shift we have cloned the translocated c-myc allele from Burkitt's lymphoma cell line BL60, which is characterized by several point mutations. The mutated c-myc allele of BL60 was stably introduced into baby hamster kidney and Burkitt's lymphoma cells. S1 nuclease and RNAase protection mapping experiments demonstrated that the mutated c-myc allele was expressed at a low level and with a normal promoter usage (P2 greater than P1) in Burkitt's lymphoma and baby hamster kidney cells. Furthermore, we have studied the expression of a construct consisting of the mutated c-myc allele, part of the bvr1 (Burkitt's variant rearranging region 1) locus, the human immunoglobulin kappa constant region, and the kappa intron enhancer after stable transfection into Burkitt's lymphoma cells. Although c-myc expression was about fivefold increased, the transcripts still initiated predominantly at promoter P2. This indicates that 5 kb of the constant kappa light-chain locus including the kappa intron enhancer is not sufficient to induce the Burkitt's lymphoma-specific promoter shift.

Animals↗
PubMed1990

Dietary-induced pre-translational control of rat fatty acid synthase.

We have examined the effects of starvation, normal lab chow and low-fat carbohydrate-rich diet on rat fatty acid synthase (FAS, EC 2.3.1.85). Under each of the dietary conditions the amount of FAS mRNA is different, the most being produced after a low-fat carbohydrate-rich diet. There is also an increase in the amount of FAS protein under the same conditions. To complete the picture we determined the incorporation of [14C]acetate into palmitate as a measure of enzyme activity. Data for cardiac and renal tissue also reflect a dietary influence on FAS. Therefore FAS mRNA, FAS protein and FAS activity are all responsive to dietary-induced signals, and our results suggest a pre-translational regulation.

Animals↗
PubMed1989

Rat mammary gland fatty acid synthase: localization of the constituent domains and two functional polyadenylation/termination signals in the cDNA.

The rat fatty acid synthase (FAS) is active only as a dimer, although the eight component functions are contained in a single polypeptide chain. Using mRNA from lactating rat mammary glands a cDNA expression library was established. With the overlapping immunologically positive clones we have an 8.9kb cDNA sequence for rat FAS. In the 3'-nontranslated region of the rat FAS cDNA we find a prototype polyadenylation/termination signal and 779 nucleotides upstream, a mutated one. Both of these polyadenylation/termination signals are used and give rise to two equally abundant mRNA species which are coordinately regulated. In the derived amino acid sequence we could locate six of the eight component functions; their order is NH2- beta-ketoacyl synthase - acetyl/malonyl transferases -enoyl reductase - acyl carrier protein - thioesterase -COOH. Comparison of FAS from different sources shows that the primary sequence is conserved only for the active residues and the amino acids in their immediate vicinity.

Amino Acid Sequence↗
PubMed1989

Structural analysis of proviral DNA in simian foamy virus (LK-3)-infected cells.

Proviral DNA of the T-cell lymphotropic simian foamy virus strain LK-3 was characterized. In infected cells, multiple copies of unintegrated linear duplex viral DNA of about 13 kbp length are present. Nuclease S1 treatment of the DNA generated two fragments of 6.5 and 6.0 kbp length that were cloned in phage and plasmid vectors. The proviral DNA contains a single-stranded gap of 109 nucleotides. A DNA fragment spanning the gap was cloned after completing the double strand by DNA synthesis in vitro. At the 3' end, the gap contains a polypurine tract (PPT) similar to the putative initiation site of retroviral plus strand DNA synthesis, suggesting discontinuous DNA synthesis. Further analysis of the genome architecture revealed LTRs of 1.7 kbp length. An additional 1.7 kbp DNA fragment was detected after nuclease S1 digestion of proviral DNA and probably represents trimmed intermediates of "strong-stop" DNA.

Animals↗
PubMed1988

Heterogeneity of primate foamy virus genomes. Brief report.

DNA of the T-lymphotropic simian foamy virus (SFV) LK-3 was cloned in a plasmid vector and used as a probe in comparative DNA:DNA hybridization studies with primate foamy viruses (SFV serotypes 1, 2, 3, 5, 6, 7, 8, fresh SFV isolates from 12 African green monkeys and one rhesus monkey, as well as human syncytium forming virus). All freshly isolated viruses seemed to be variants of SFV types 2 and 3. Especially LK-3 appeared to be closely related to SFV type 3. No hybridization of the LK-3 clone was observed to DNA sequences of human immunodeficiency virus (HIV-1).

Animals↗
PubMed1987

Identification and sequencing of cDNA clones for the rodent negative acute-phase protein alpha 1-inhibitor 3.

Rat alpha 1-inhibitor 3 clones were isolated by immunological screening of a lambda gt11 cDNA library prepared from rat liver poly(A)-rich RNA. The recombinant cDNA clones were identified by the absence of their immunoprecipitable products following hybrid-arrested in vitro translation. The size of the cognate poly(A)-rich RNA was estimated to be roughly 5000 residues. Approximately 16 h after induction of inflammation the amount of alpha 1-inhibitor 3 poly(A)-rich RNA decreases as shown by dot-blot hybridization and Northern analyses. The response of this negative acute-phase plasma protein to inflammation may therefore be considered to be at the pretranslational level. The characterized DNA constitutes an open reading frame of 225 amino acids followed by a canonical eucaryotic polyadenylation signal and a poly(A) tail. Sequence microheterogeneity, particularly in the 3'-flanking region was observed. An amino acid homology of 70% for alpha 1-inhibitor 3 with human and rodent alpha 2-macroglobulin emphasizes the evolutionary relationship of the macroglobulins.

Acute-Phase Proteins↗
PubMed1986

The pentafunctional FAS1 gene of yeast: its nucleotide sequence and order of the catalytic domains.

FAS1, the structural gene of the pentafunctional fatty acid synthetase subunit beta in Saccharomyces cerevisiae has been sequenced. Its reading frame represents an intron-free nucleotide sequence of 5,535 base pairs, corresponding to a protein of 1,845 amino acids with a molecular weight of 205,130 daltons. In addition to the coding sequence, 1,468 base pairs of its 5'-flanking region were determined. S1 nuclease mapping revealed two transcriptional initiation sites; 5 and 36 base pairs upstream of the translational start codon. Within the flanking sequences two TATATAAA boxes, several A-rich and T-rich blocks and a TAG...TATGTT...TATGTT...TTT sequence were found and are discussed as transcriptional initiation and termination signals, respectively. The order of catalytic domains in the cluster gene was established by complementation of defined fas1 mutants with overlapping FAS1 subclones. Acetyl transferase (amino acids 1-468) is located proximal to the N-terminus of subunit beta, followed by the enoyl reductase (amino acids 480-858), the dehydratase (amino acids 1,134-1,615) and the malonyl/palmityl transferase (amino acids 1,616-1,845) domains. One major inter-domain region of about 276 amino acids with so far unknown function was found between the enoyl reductase and dehydratase domains. The substrate-binding serine residues of acetyl, malonyl and palmityl transferases were identified within the corresponding domains. Significant sequence homologies exist between the acyl transferase active sites of yeast and animal fatty acid synthetases. Similarly, a putative sequence of the enoyl reductase active site was identified.

Amino Acid Sequence↗
PubMed1986

Detection and characterization of infectious DNA intermediates of a primary foamy virus.

DNA from human T-lymphoid (Molt-4) and hamster kidney (BHK-21) cells infected with the T-lymphotropic simian foamy virus LK-3 was shown to be infectious, when assayed by transfection of BHK-21 cells. The proviral genome was further characterized by blot hybridization to a specific cDNA probe, which had been prepared by reverse transcription in vitro using viral RNA and RNA-dependent DNA polymerase present in cytoplasmic extracts of infected BHK-21 cells. This probe hybridized to a DNA species of 14 kbp in extracts from LK-3-infected diploid human fibroblasts, Molt-4 and BHK-21 cells, whereas no hybridization occurred with DNA from the respective uninfected controls. No integrated proviral DNA could be demonstrated, and the 14 kbp DNA was shown not to represent circular DNA. The patterns of restriction endonuclease and S1 nuclease fragments indicated a unique configuration of linear double-stranded DNA containing a single-stranded section separating two subunits one of which may be sufficient to transmit LK-3 by transfection with DNA.

Animals↗
PubMed1984

Molecular cloning of the yeast fatty acid synthetase genes, FAS1 and FAS2: illustrating the structure of the FAS1 cluster gene by transcript mapping and transformation studies.

From a Saccharomyces cerevisiae gene bank contained in the novel yeast cosmid shuttle vector pMS201 the fatty acid synthetase (FAS) genes FAS1 and FAS2 were isolated. FAS clones were identified by in situ colony hybridization using two yeast DNA probes apparently capable of producing avian FAS cross-reacting material (J. Carbon, personal communication). Classification as FAS1 or FAS2 clones was achieved by their specific transformation of fas1 and fas2 yeast mutants. By transcription mapping FAS1 was assigned to about 5.3 kb within 14.8 kb of chromosomal DNA covered by two genomically adjacent BamHI fragments. The FAS2 gene was localized on a single BamHI fragment of 25 kb. One of the FAS clones ( FAS2 ) produces immunologically cross-reacting material in Escherichia coli. High frequency transformation of fas1 mutants was only observed with one subclone, pMS3021 , containing the intact FAS1 locus. Other DNA segments cloned in the same self-replicating vector but representing only part of FAS1 exhibited drastically lower transformation rates. As evident from this and from FAS1 /TRP1-cotransformation rates only the intact FAS1 gene in pMS3021 is capable of fas1 -mutant complementation. With partial FAS1 genes, even when coding for an intact equivalent of the mutated domain, their chromosomal integration is necessary for the expression of FAS. In integrative transformants the coexistence of integrated and autonomously replicating plasmid DNA was demonstrated. Both, the extrachromosomal and chromosomally integrated FAS DNA was mitotically unstable. Transformation studies using subcloned FAS1 DNA segments revealed the relative locations of the enoyl reductase and dehydratase domains within this pentafunctional cluster gene.

Cloning, Molecular↗
PubMed1981

Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1+ regulatory gene.

The early reactions in the catabolism of quinic acid in Neurospora crassa are controlled by at least four genes which are clustered on linkage group VII. Three of the loci (qa-2, qa-4, and qa-3) encode enzymes that convert quinic acid to protocatechuic acid. The fourth gene (qa-1) encodes a positive regulatory protein which, in the presence of quinic acid, leads to the de novo synthesis of the other proteins in the qa cluster. This communication describes a series of recombinant plasmids that span 36.5 kilobases of linkage group VII and contain the coding sequences for qa-2, qa-4, qa-3, and the qa-1 regulatory protein. The plasmids were obtained by partial digestion of wild-type N. crassa DNA with EcoRI and ligation into the cosmid cloning vehicle pHC79. Two independently derived plasmids (pMSK331 and pMSK335), each containing 36.5-kilobase inserts, were shown by transformation back into N. crassa to contain the entire qa gene cluster. A preliminary physical organization of the gene cluster is presented. An improved procedure for the transformation of N. crassa with plasmid DNA is also described.

DNA Restriction Enzymes↗
PubMed1981

Genetic organization and transcriptional regulation in the qa gene cluster of Neurospora crassa.

A transcription map of the qa gene cluster of Neurospora crassa has been constructed by using cloned DNA fragments as hybridization probes. The mRNAs encoded in the previously identified qa-2 (3-dehydroquinate hydro-lyase, EC 4.2.1.10), qa-4 (dehydroshikimate dehydratase), qa-3 (quinate:NAD+ 3-oxidoreductase, EC 1.1.1.24), and qa-1 (regulatory protein) genes have been characterized. In addition, mRNAs encoded in two new genes in this cluster (qa-x, qa-y) have been identified. Regulation of this system occurs at the level of transcription and is under the combined control of quinic acid and the qa-1 protein. The qa cluster represents a group of adjacent coding sequences which occupy approximately 18 kilobases in linkage group VII. The expression of the qa-1 gene appears to be constitutive but also autoregulated. mRNAs encoded in genes flanking the qa gene cluster have also been identified.

Gene Expression Regulation↗
PubMed1979

Constitutive expression in Escherichia coli of the Neurospora crassa structural gene encoding the inducible enzyme catabolic dehydroquinase.

In Neurospora crassa the qa-2 gene, which encodes catabolic dehydroquinase, is under positive control exerted by the inducer quinic acid and an activator protein encoded in the closely linked qa-1 gene. In order to determine if this regulatory mechanism is maintained when the qa-2 gene is cloned on a recombinant plasmid and expressed in Escherichia coli, molecular cloning experiments have been performed using DNA isolated from a qa-1+ (inducible), a qa-1C (constitutive) and two qa-1 (non-inducible) strains of N. crassa. The results demonstrate that the level of expression of the qa-2 gene in E. coli is completely independent of the mutational state of the qa-1 gene. Moreover, the level of expression of the cloned qa-2 gene was unaffected by either an intracellularly produced inducer of catabolic dehydroquinase or by the general procaryotic positive effector, the CAP factor. The weight of evidence thus supports the conclusion that transcription of the N. crassa qa-2 gene in E. coli does not require the qa-1 activator protein and thus is not controlled by the same mecahnism which functions in N. crassa.

Centrifugation, Density Gradient↗
PubMed1979

Exclusion of VHa and VHy loci expression on individual B cells from normal and VH allotype-suppressed rabbits.

The distribution of two heavy chain subgroups, VHa and VHy, on rabbit peripheral blood lymphocytes was examined by double membrane immunofluorescence. Fluroescent anti-a1 and anti-y33 were found to react with separate B cell populations; no doubly stained cells were observed. Further evidence for the independent expression of genes controlling the VHa and VHy subgroups were obtained by neonatal suppression of a 2 or y33 in a2y33/a3y- heterozygous rabbits. Suppression of VHa did not affect the expression of VHy, nor did the suppression of VHy affect the expression of VHa. The expression of a single VH gene per B cell is in marked contrast to the simultaneous expression of multiple CH genes.

Aging↗