scholarly journals Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.

1990 ◽  
Vol 10 (6) ◽  
pp. 2638-2644 ◽  
Author(s):  
S Marathe ◽  
I F Connerton ◽  
J R Fincham
Keyword(s):  
Amino Acid ◽  
Carbon Source ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Protein Product ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
Reading Frame ◽  
Acetate Utilization ◽  
Group 2

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.

Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product

1990 ◽  
Vol 10 (6) ◽  
pp. 2638-2644
Author(s):  
S Marathe ◽  
I F Connerton ◽  
J R Fincham
Keyword(s):  
Amino Acid ◽  
Carbon Source ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Protein Product ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
Reading Frame ◽  
Acetate Utilization ◽  
Group 2

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.

scholarly journals Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

Genetics ◽  
1992 ◽  
Vol 131 (3) ◽  
pp. 531-539 ◽  
Author(s):  
C Bornaes ◽  
J G Petersen ◽  
S Holmberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Open Reading Frame ◽  
Predicted Amino Acid Sequence ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Threonine Dehydratase ◽  
Nuclease Mapping

Abstract The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

scholarly journals Cloning of pig prostaglandin F2alphaFP receptor cDNA and expression of its mRNA in the corpora lutea

Reproduction ◽  
2003 ◽  
pp. 53-64 ◽  
Author(s):  
U Boonyaprakob ◽  
JE Gadsby ◽  
V Hedgpeth ◽  
P Routh ◽  
GW Almond
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Oestrous Cycle ◽  
Small Cells ◽  
Predicted Amino Acid Sequence ◽  
Corpora Lutea ◽  
Reading Frame ◽  
Luteal Tissue ◽  
Fp Receptor ◽  
Steroidogenic Cells

Changes in the expression and localization of luteal mRNA for PGF(2alpha) (FP) receptors may be critical in determining the luteolytic action of PGF(2alpha) in pig corpora lutea. In this study, a full-length FP receptor (FPr) cDNA was isolated and cloned from pig corpora lutea. This isolate (GenBank accession no. U91520) contains an open reading frame of 1086 bases coding for a protein of 362 amino acids with seven potential transmembrane domains. The predicted amino acid sequence of this isolate was 83% identical to the FPr amino acid sequence of other species including sheep, cattle and humans. Northern blot analysis showed the presence of an FPr message of about 5 kb in mRNA from pig corpora lutea. Relatively weak FPr mRNA expression was detected on day 4 and day 7 of the oestrous cycle. The expression was greater (P < 0.05) on days 10, 13 and 15 than on days 4 and 7. In situ hybridization analysis revealed that mRNA for FPr was expressed predominantly in the steroidogenic large luteal subtype of cell, although there was some expression in small luteal cells, with histological appearance of steroidogenic small cells. Localization of hybridization signals of FPr was observed in luteal tissue at all stages examined. These data demonstrate that FPr is expressed in pig corpora lutea throughout the oestrous cycle and that upregulation of the FPr mRNA occurs when the corpora lutea becomes sensitive to PGF(2alpha). Direct luteal targets of PGF(2alpha) appear to be primarily large steroidogenic cells in this species.

scholarly journals Insulin regulation of a novel WD-40 repeat protein in adipocytes

2001 ◽  
Vol 168 (2) ◽  
pp. 325-332 ◽  
Author(s):  
BD Rodgers ◽  
MA Levine ◽  
M Bernier ◽  
C Montrose-Rafizadeh
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tissue Expression ◽  
Northern Blotting ◽  
Predicted Amino Acid Sequence ◽  
Mrna Levels ◽  
Degenerate Primers ◽  
Hek 293 ◽  
Reading Frame

A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.

scholarly journals The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae.

1996 ◽  
Vol 132 (4) ◽  
pp. 549-563 ◽  
Author(s):  
E E Swartzman ◽  
M N Viswanathan ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Amino Acid ◽  
Carbon Source ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Atp Binding ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
Atp Binding Cassette

The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).

scholarly journals Peroxisomal multifunctional enzyme of β-oxidation metabolizing d-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization

1997 ◽  
Vol 321 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Yong-Mei QIN ◽  
Matti H. POUTANEN ◽  
Heli M. HELANDER ◽  
Ari-Pekka KVIST ◽  
Kirsi M. SIIVARI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rat Liver ◽  
Predicted Amino Acid Sequence ◽  
Peptide Fragments ◽  
Reading Frame ◽  
Multifunctional Enzyme ◽  
Type Iv ◽  
Liver Peroxisomes ◽  
Enoyl Coa Hydratase

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an Mr of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578–21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted Mr of 79331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17β-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal β-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase–hydratase–dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of β-oxidation but through reciprocal stereochemical courses.

scholarly journals L-Pipecolic acid oxidase, a human enzyme essential for the degradation of L-pipecolic acid, is most similar to the monomeric sarcosine oxidases

2000 ◽  
Vol 345 (3) ◽  
pp. 487-494 ◽  
Author(s):  
Gabriele DODT ◽  
Do G. KIM ◽  
Sylvia A. REIMANN ◽  
Bernadette E. REUBER ◽  
Katherine MCCABE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Reverse Genetics ◽  
Human Fibroblasts ◽  
Protein Product ◽  
Pipecolic Acid ◽  
Acid Oxidase ◽  
Peroxisomal Enzyme ◽  
Reading Frame

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, we cloned the associated human gene and expressed its protein product. The cDNA was cloned with the use of a reverse genetics approach based on the amino acid sequence obtained from purified L-pipecolic acid oxidase from monkey. The complete cDNA, obtained by conventional library screening and 5ʹ rapid amplification of cDNA ends, encompassed an open reading frame of 1170 bases, translating to a 390-residue protein. The translated protein terminated with the sequence AHL, a peroxisomal targeting signal 1. Indirect immunofluorescence studies showed that the protein product was expressed in human fibroblasts in a punctate pattern that co-localized with the peroxisomal enzyme catalase. A BLAST search with the amino acid sequence showed 31% identity and 53% similarity with Bacillus sp. NS-129 monomeric sarcosine oxidase, as well as similarity to all sarcosine oxidases and dehydrogenases. No similarity was found to the peroxisomal D-amino acid oxidases. The recombinant enzyme oxidized both L-pipecolic acid and sarcosine. However, PBD patients who lack the enzyme activity accumulate only L-pipecolic acid, suggesting that in humans in vivo, this enzyme is involved mainly in the degradation of L-pipecolic acid.

scholarly journals Molecular Characterization of the β-N-Acetylglucosaminidase of Escherichia coliand Its Role in Cell Wall Recycling

2000 ◽  
Vol 182 (17) ◽  
pp. 4836-4840 ◽  
Author(s):  
Qiaomei Cheng ◽  
Hongshan Li ◽  
Keith Merdek ◽  
James T. Park
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sequence Data ◽  
Degradation Products ◽  
Predicted Amino Acid Sequence ◽  
The Novel ◽  
Sequence Comparisons ◽  
Reading Frame ◽  
Vibrio Furnissii

ABSTRACT The β-N-acetylglucosaminidase of Escherichia coli was found to have a novel specificity and to be encoded by a gene (nagZ) that maps at 25.1 min. It corresponds to an open reading frame, ycfO, whose predicted amino acid sequence is 57% identical to that of Vibrio furnissiiExoII. NagZ hydrolyzes the β-1,4 glycosidic bond betweenN-acetylglucosamine and anhydro-N-acetylmuramic acid in cell wall degradation products following their importation into the cell during the process for recycling cell wall muropeptides. From amino acid sequence comparisons, the novel β-N-acetylglucosaminidase appears to be conserved in all 12 gram-negative bacteria whose complete or partial genome sequence data are available.

scholarly journals Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing.

1988 ◽  
Vol 8 (3) ◽  
pp. 1113-1122 ◽  
Author(s):  
E Czarnecka ◽  
R T Nagao ◽  
J L Key ◽  
W B Gurley
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Stress Protein ◽  
Initiation Site ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
Soybean Seedlings ◽  
Nuclease Mapping

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.

scholarly journals Complete nucleotide sequence of cDNA and predicted amino acid sequence of rat acyl-CoA oxidase.

1987 ◽  
Vol 262 (17) ◽  
pp. 8131-8137 ◽  
Author(s):  
S Miyazawa ◽  
H Hayashi ◽  
M Hijikata ◽  
N Ishii ◽  
S Furuta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Complete Nucleotide Sequence ◽  
Predicted Amino Acid Sequence ◽  
Acyl Coa Oxidase
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