scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1711-1721
Author(s):  
E M McIntosh ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Hindiii Site ◽  
Reading Frame ◽  
Hindiii Restriction Site ◽  
Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Nucleotide and Amino Acid Sequences of the Metallo-β-Lactamase, ImiS, from Aeromonas veronii bv. sobria

1998 ◽  
Vol 42 (2) ◽  
pp. 436-439 ◽  
Author(s):  
T. R. Walsh ◽  
W. A. Neville ◽  
M. H. Haran ◽  
D. Tolson ◽  
D. J. Payne ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Aeromonas Veronii ◽  
Putative Active Site ◽  
Lactamase Gene ◽  
Active Site Sequence

ABSTRACT The Aeromonas veronii bv. sobria metallo-β-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-β-lactamases.

scholarly journals Sequence of the Structural Gene for Xanthine Dehydrogenase (rosy Locus) in Drosophila melanogaster

Genetics ◽  
1987 ◽  
Vol 116 (1) ◽  
pp. 67-73
Author(s):  
Tim P Keith ◽  
Margaret A Riley ◽  
Martin Kreitman ◽  
R C Lewontin ◽  
Daniel Curtis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Structural Gene ◽  
Xanthine Dehydrogenase ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Eco Ri

ABSTRACT We determined the nucleotide sequence of a 4.6-kb Eco RI fragment containing 70% of the rosy locus. In combination with information on the 5′ sequence, the gene has been sequenced in entirety. rosy cDNAs have been isolated and intron/exon boundaries have been determined. We find an open reading frame which spans four exons and would encode a protein of 1335 amino acids. The molecular weight of the encoded protein (xanthine dehydrogenase), based on the amino acid translation, is 146,898 daltons which agrees well with earlier biophysical estimates. Characteristics of the protein are discussed.

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 Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (5) ◽  
pp. 1711-1721 ◽  
Author(s):  
E M McIntosh ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Hindiii Site ◽  
Reading Frame ◽  
Hindiii Restriction Site ◽  
Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Primary structure of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (12) ◽  
pp. 2837-2842
Author(s):  
K A Krzywicki ◽  
M C Brandriss
Keyword(s):  
Rna Synthesis ◽  
Signal Sequence ◽  
Mitochondrial Pathway ◽  
Open Reading Frame ◽  
Amino Terminus ◽  
Transcript Mapping ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Proline Utilization

The PUT2 gene, believed to encode delta 1-pyrroline-5-carboxylate dehydrogenase, has been completely sequenced. The DNA contains an open reading frame of 1,725 base pairs encoding a protein of 575 amino acids. Transcript mapping with both S1 nuclease and primer extension methods revealed numerous initiation sites of RNA synthesis 50 to 80 base pairs downstream from several TATA boxes. The deduced amino acid sequence of delta 1-pyrroline-5-carboxylate dehydrogenase contains a highly basic amino terminus that may serve as the signal sequence that targets this protein to the mitochondrion.

scholarly journals Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999 ◽  
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

scholarly journals Cloning of a Mucin-Desulfating Sulfatase Gene fromPrevotella Strain RS2 and Its Expression Using aBacteroides Recombinant System

2000 ◽  
Vol 182 (11) ◽  
pp. 3002-3007 ◽  
Author(s):  
Damian P. Wright ◽  
Catriona G. Knight ◽  
Shanthi G. Parkar ◽  
David L. Christie ◽  
Anthony M. Roberton
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Signal Sequence ◽  
Active Form ◽  
Open Reading Frame ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein

ABSTRACT A gene encoding the mucin-desulfating sulfatase inPrevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into aBacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

scholarly journals DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species

1986 ◽  
Vol 6 (1) ◽  
pp. 265-276
Author(s):  
C Upton ◽  
G McFadden
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Homology ◽  
Inverted Repeat ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cesium Chloride ◽  
Open Reading Frame ◽  
Terminal Inverted Repeat ◽  
Reading Frame

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.

scholarly journals Primary structure of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (12) ◽  
pp. 2837-2842 ◽  
Author(s):  
K A Krzywicki ◽  
M C Brandriss
Keyword(s):  
Rna Synthesis ◽  
Signal Sequence ◽  
Mitochondrial Pathway ◽  
Open Reading Frame ◽  
Amino Terminus ◽  
Transcript Mapping ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Proline Utilization

The PUT2 gene, believed to encode delta 1-pyrroline-5-carboxylate dehydrogenase, has been completely sequenced. The DNA contains an open reading frame of 1,725 base pairs encoding a protein of 575 amino acids. Transcript mapping with both S1 nuclease and primer extension methods revealed numerous initiation sites of RNA synthesis 50 to 80 base pairs downstream from several TATA boxes. The deduced amino acid sequence of delta 1-pyrroline-5-carboxylate dehydrogenase contains a highly basic amino terminus that may serve as the signal sequence that targets this protein to the mitochondrion.

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