scholarly journals Enhancement of Secretion and Extracellular Stability of Staphylokinase in Bacillus subtilis bywprA Gene Disruption

2000 ◽  
Vol 66 (2) ◽  
pp. 476-480 ◽  
Author(s):  
Sang Jun Lee ◽  
Dong Min Kim ◽  
Kwang Hee Bae ◽  
Si Myung Byun ◽  
Jae Hoon Chung
Keyword(s):  
Bacillus Subtilis ◽  
Gene Disruption ◽  
Therapeutic Potential ◽  
Signal Sequence ◽  
Open Reading Frame ◽  
Expression Plasmid ◽  
Reading Frame ◽  
Extracellular Milieu ◽  
Foreign Proteins

ABSTRACT Staphylokinase (SAK), a polypeptide secreted byStaphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.

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.

Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure

1996 ◽  
Vol 16 (1) ◽  
pp. 73-80 ◽  
Author(s):  
D A Rubin ◽  
J H Youson ◽  
L E Marra ◽  
R M Dores
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Leader Sequence ◽  
Open Reading Frame ◽  
Russian Sturgeon ◽  
Reading Frame ◽  
Terminal Codon

ABSTRACT A cDNA containing the sequence of GH was cloned and sequenced from a pituitary cDNA library for the holostean fish Lepisosteus osseus (common name: gar). The gar GH cDNA contained an open reading frame of 633 nucleotides and a 3′ untranslated region (including the terminal codon TAG) of 1058 nucleotides. The overall length of the gar GH cDNA including leader sequence, signal sequence, hormone sequence and 3′ untranslated region was 1713 nucleotides. Thus, the gar GH cDNA is the largest vertebrate GH cDNA yet cloned. A comparison of GH sequences from ancient (holostean fishes — gar and bowfin; one chondrostean fish — the Russian sturgeon) and more modern (27 species of teleosts) members of class Actinopterygii indicate that members of this class have maintained many of the invariant residues deemed necessary for GH folding motifs (intramolecular relationships) observed in mammals.

scholarly journals One of the tightly clustered genes of the mouse surfeit locus is a highly expressed member of a multigene family whose other members are predominantly processed pseudogenes.

1988 ◽  
Vol 8 (9) ◽  
pp. 3898-3905 ◽  
Author(s):  
C Huxley ◽  
T Williams ◽  
M Fried
Keyword(s):  
Multigene Family ◽  
Open Reading Frame ◽  
The Other ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Reading Frame ◽  
Processed Pseudogenes ◽  
Dna Fragment ◽  
Basic Polypeptide

The mouse surfeit locus is unusual in that it contains a number of closely clustered genes (Surf-1, -2, and -4) that alternate in their direction of transcription (T. Williams, J. Yon, C. Huxley, and M. Fried, Proc. Natl. Acad. Sci. USA 85:3527-3530, 1988). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by 15 to 73 base pairs (bp), and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp (T. Williams and M. Fried, Mol. Cell. Biol. 6:4558-4569, 1986; T. Williams and M. Fried, Nature (London) 322:275-279, 1986). A fourth gene in this locus, Surf-3, which is a member of a multigene family, has been identified. The poly(A) addition site of Surf-3 lies only 70 bp from the poly(A) addition site of Surf-1. Transcription of Surf-3 has been studied in the absence of the other members of its multigene family after transfection of a cloned genomic mouse DNA fragment, containing the Surf-3 gene, into heterologous monkey cells. Surf-3 specifies a highly expressed 1.0-kilobase mRNA that contains a long open reading frame of 266 amino acids, which would encode a highly basic polypeptide (23% Arg plus Lys). The other members of the Surf-3 multigene family are predominantly, if not entirely, intronless pseudogenes with the hallmarks of being generated by reverse transcription. The role of the very tight clustering on regulation of expression of the genes in the surfeit locus is discussed.

scholarly journals Role of a Cytotoxic-T-Lymphocyte Epitope-Defined, Alternative gag Open Reading Frame in the Pathogenesis of a Murine Retrovirus-Induced Immunodeficiency Syndrome

2005 ◽  
Vol 79 (7) ◽  
pp. 4308-4315 ◽  
Author(s):  
Arti Gaur ◽  
William R. Green
Keyword(s):  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Virus ◽  
Protein S ◽  
Initiation Site ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Ctl Epitope ◽  
Immunodeficiency Syndrome

ABSTRACT LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60 gag ). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.

Role of TlyA in the Biology of Uncultivable Mycobacteria

2022 ◽  
Vol 25 ◽  
Author(s):  
Mukul Sharma ◽  
Pushpendra Singh
Keyword(s):  
Comparative Analysis ◽  
Evolutionary Biology ◽  
Signal Sequence ◽  
Transmembrane Segment ◽  
Structural Characterisation ◽  
Extracellular Milieu ◽  
Transmembrane Segments ◽  
Virulence Protein ◽  
Extracellular Environment

Abstract: TlyA proteins are related to distinct functions in a diverse spectrum of bacterial pathogens including mycobacterial spp. There are several annotated proteins function as hemolysin or pore forming molecules that play an important role in the virulence of pathogenic organisms. Many studies reported the dual activity of mycobacterial TlyA as ‘hemolysin’ and ‘S-adenosylmethionine dependent rRNA methylase’. To act as a hemolysin, a sequence must have a signal sequence and transmembrane segment which helps the protein to enter the extracellular environment. Interestingly, the mycobacterial tlyA has neither a traditional signal sequences of general/sec/tat pathways nor any transmembrane segments are present. Still it can reach the extracellular milieu with the help of non-classical signal mechanisms. Also, retention of tlyA in cultivable mycobacterial pathogens (such as Mycobacterium tuberculosis and M. marinum) as well as uncultivated mycobacterial pathogens despite their extreme reductive evolution (such as M. leprae, M. lepromatosis and M. uberis) suggests its crucial role in evolutionary biology of pathogenic mycobacteria. Numerous virulence factors have been characterised from the uncultivable mycobacteria but the information of TlyA protein is still limited in terms of molecular and structural characterisation. The genomic insights offered by comparative analysis of TlyA sequences and its conserved domains reveal its pore forming activity which further confirms its role as a virulence protein, particularly in uncultivable mycobacteria. Therefore, this review presents a comparative analysis of mycobacterial TlyA family by sequence homology and alignment to improve our understanding of this unconventional hemolysin and RNA methyltransferase TlyA of uncultivable mycobacteria.

Controlled atmospheres and sugar can delay malate synthase gene expression during asparagus senescence

2002 ◽  
Vol 29 (9) ◽  
pp. 1045 ◽  
Author(s):  
Simon A. Coupe ◽  
Ben K. Sinclair ◽  
Sheryl D. Somerfield ◽  
Paul L. Hurst
Keyword(s):  
Gene Expression ◽  
Single Gene ◽  
Malate Synthase ◽  
Open Reading Frame ◽  
Asparagus Officinalis ◽  
High Identity ◽  
Reading Frame ◽  
Controlled Atmospheres ◽  
Foliar Senescence

A cDNA clone encoding malate synthase (MS; EC 4.1.3.2) was isolated from a 48-h postharvest asparagus (Asparagus officinalis L.) spear cDNA library using a MS clone from Brassica napus. The asparagus MS (AoMS1) cDNA hybridized to a 1.9-kb transcript that increased in abundance preferentially in spear-tip tissue during postharvest storage. The AoMS1 transcript also accumulated during natural foliar senescence of asparagus fern. The cDNA consists of 1960 nucleotides with an open reading frame of 1665 nucleotides or 555 amino acids, and encodes a deduced protein with a predicted Mr of 63 kDa and a pI of 8.1. The deduced amino acid sequence of AoMS1 showed high identity with the B. napus MS clone (77.2%) used to isolate it, and with MS from cucumber (77%). Genomic Southern analysis suggests that a single gene in asparagus encodes AoMS1. Controlled- atmosphere treatments aimed at reducing deterioration of harvested asparagus spears reduced the expression of AoMS1. The reduction was correlated with the reduced oxygen level, and reduced MS enzyme activity was also observed. Asparagus cell cultures were used to test the role of sugar status in regulating AoMS1 gene expression. In cultures without sucrose there was an accumulation of AoMS1 transcript that was absent in cultures containing sucrose.

scholarly journals Mutation in yaaT Leads to Significant Inhibition of Phosphorelay during Sporulation in Bacillus subtilis

2002 ◽  
Vol 184 (20) ◽  
pp. 5545-5553 ◽  
Author(s):  
Shigeo Hosoya ◽  
Kei Asai ◽  
Naotake Ogasawara ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Response Regulator ◽  
Significant Inhibition ◽  
Open Reading Frame ◽  
Histidine Kinases ◽  
Reading Frame ◽  
Green Fluorescent ◽  
Sporulation Genes

ABSTRACT In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of σH, which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

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