scholarly journals The exuT Gene of Erwinia chrysanthemi EC16: Nucleotide Sequence, Expression, Localization, and Relevance of the Gene Product

1998 ◽  
Vol 11 (4) ◽  
pp. 270-276 ◽  
Author(s):  
B. J. Haseloff ◽  
T. L. Freeman ◽  
V. Valmeekam ◽  
M. W. Melkus ◽  
F. Oner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Membrane Protein ◽  
Plant Cell Wall ◽  
Amino Acid Content ◽  
Immunoblot Analysis ◽  
Integral Membrane Protein ◽  
Erwinia Chrysanthemi ◽  
Nucleotide Sequence Analysis ◽  
Tissue Maceration

Galacturonic acid (GalUA) is a major component of pectin and polygalacturonic acid in the plant cell wall. In the phytopathogen Erwinia chrysanthemi, the uptake of molecules derived from degradation of these polymers is an important early step in the events preceding induction of pectinases, ultimately leading to plant tissue maceration. Uptake systems for GalUA and dimers of GalUA have been described and shown to be inducible in E. chrysanthemi. The GalUA uptake gene (exuT) was cloned and sequenced. Nucleotide sequence analysis identified an open reading frame encoding a 345-amino-acid polypeptide with a calculated mass of 37,825 Da. This polypeptide is predicted to be an integral membrane protein based on its high nonpolar amino acid content and hydropathic profile. Localization studies with the labeled polypeptide in the T7-RNA polymerase system also suggest that ExuT is a membrane protein. This evidence is further supported by the observation of hybrid ExuT-PhoA proteins in the bacterial cytoplasmic membrane following immunoblot analysis. Northern (RNA) analysis indicated that the gene is inducible in the presence of the monomer, GalUA. A targeted mutation in the exuT gene affected the utilization of GalUA as a sole carbon source for growth. Maceration of potato tuber tissue by this mutant was delayed and reduced, when compared with the parental strain EC16.

Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (11) ◽  
pp. 3024-3034
Author(s):  
E T Young ◽  
D Pilgrim
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Alcohol Dehydrogenase ◽  
Nuclear Gene ◽  
Amino Acid Identity ◽  
Leader Peptide ◽  
Nucleotide Sequence Analysis ◽  
Amino Terminal ◽  
Cloned Gene

The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not

1987 ◽  
Vol 7 (6) ◽  
pp. 2173-2179
Author(s):  
P C Yelick ◽  
R Balhorn ◽  
P A Johnson ◽  
M Corzett ◽  
J A Mazrimas ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Protamine 1 ◽  
Protamine 2

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

Cloning and nucleotide sequence of cDNA encoding human erythrocyte band 7 integral membrane protein

1991 ◽  
Vol 1090 (1) ◽  
pp. 123-128 ◽  
Author(s):  
Christine M. Hiebl-Dirschmied ◽  
Barbara Entler ◽  
Claudia Glotzmann ◽  
Ingrid Maurer-Fogy ◽  
Christian Stratowa ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Membrane Protein ◽  
Human Erythrocyte ◽  
Integral Membrane Protein

scholarly journals Identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (VAP-33): a broadly expressed protein that binds to VAMP

1998 ◽  
Vol 333 (2) ◽  
pp. 247-251 ◽  
Author(s):  
M. Lynn WEIR ◽  
Amira KLIP ◽  
William S. TRIMBLE
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Membrane Protein ◽  
Structural Organization ◽  
Aplysia Californica ◽  
Amino Acid Identity ◽  
Accession Number ◽  
Human Homologue ◽  
Specific Expression ◽  
Acid Identity

We report the identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein (hVAP-33) that has been implicated in neuronal exocytosis in Aplysia californica. This hVAP-33 shared 50% amino acid identity with the A. californica form and had similar length, structural organization and VAMP-binding abilities. However, in contrast with the neuron-specific expression seen in A. californica, hVAP-33 was broadly expressed, suggesting possible roles in vesicle fusion in both neuronal and non-neuronal cells. The nucleotide sequence reported in this paper can be found in GenBank's database using accession number AF057358.

scholarly journals Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

1996 ◽  
Vol 135 (3) ◽  
pp. 585-595 ◽  
Author(s):  
M J Kuehn ◽  
R Schekman ◽  
P O Ljungdahl
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Plasma Membrane Atpase ◽  
Amino Acid Permease ◽  
Transport Vesicles ◽  
Amino Acid Permeases ◽  
Histidine Permease

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

scholarly journals The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation.

1995 ◽  
Vol 128 (3) ◽  
pp. 307-319 ◽  
Author(s):  
X Tan ◽  
H R Waterham ◽  
M Veenhuis ◽  
J M Cregg
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Hansenula Polymorpha ◽  
Methylotrophic Yeast ◽  
Integral Membrane Protein ◽  
Peroxisome Biogenesis ◽  
Primary Sequence ◽  
Zinc Finger Motifs ◽  
Molecular Machinery

We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.

scholarly journals Cloning and Molecular Characterization of Plasmid-Encoded Antigens of Borrelia burgdorferi

1999 ◽  
Vol 67 (9) ◽  
pp. 4407-4417 ◽  
Author(s):  
Jonathan T. Skare ◽  
Denise M. Foley ◽  
Santiago R. Hernandez ◽  
Deanna C. Moore ◽  
David R. Blanco ◽  
...  
Keyword(s):  
Amino Acid ◽  
Borrelia Burgdorferi ◽  
Virulent Strain ◽  
Immunoblot Analysis ◽  
Rabbit Serum ◽  
Nucleotide Sequence Analysis ◽  
Expression Library ◽  
Circular Plasmid ◽  
Amino Acid Repeats ◽  
Mammalian Infection

ABSTRACT Thirteen independent clones that encode Borrelia burgdorferi antigens utilizing antiserum from infection-immune rabbits were identified. The serum was adsorbed against noninfectiousB. burgdorferi B31 to enrich for antibodies directed against either infection-associated antigens of B. burgdorferi B31 or proteins preferentially expressed during mammalian infection. The adsorption efficiency of the immune rabbit serum (IRS) was assessed by Western immunoblot analysis with protein lysates derived from infectious and noninfectious B. burgdorferi B31. The adsorbed IRS was used to screen a B. burgdorferi expression library to identify immunoreactive phage clones. Clones were then expressed in Escherichia coli and subsequently analyzed by Western blotting to determine the molecular mass of the recombinant B. burgdorferi antigens. Southern blot analysis of the 13 clones indicated that 10 contained sequences unique to infectious B. burgdorferi. Nucleotide sequence analysis indicated that the 13 clones were composed of 9 distinct genetic loci and that all of the genes identified were plasmid encoded. Five of the clones carried B. burgdorferi genes previously identified, including those encoding decorin binding proteins A and B (dbpAB), a rev homologue present on the 9-kb circular plasmid (cp9), a rev homologue from the 32-kb circular plasmid (cp32-6), erpM, and erpX. Additionally, four previously uncharacterized loci with no known homologues were identified. One of these unique clones encoded a 451-amino-acid lipoprotein with 21 consecutive, invariant 9-amino-acid repeats near the amino terminus that we have designated VraA (for “virulent strain-associated repetitive antigen A”). Since all the antigens identified are recognized by serum from infection immune rabbits, these antigens represent potential vaccine candidates and, based on the identification of dbpAB in this screen, may also be involved in pathogenic processes operative in Lyme borreliosis.

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