Molecular cloning of extensive sequences of the in vitro synthesized chicken ovalbumin structural gene

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.

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Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro.

Journal of Bacteriology ◽

10.1128/jb.146.2.668-675.1981 ◽

1981 ◽

Vol 146 (2) ◽

pp. 668-675 ◽

Cited By ~ 71

Author(s):

H Inouye ◽

S Michaelis ◽

A Wright ◽

J Beckwith

Keyword(s):

Escherichia Coli ◽

Alkaline Phosphatase ◽

Structural Gene ◽

Restriction Mapping ◽

Deletion Mutants

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Molecular Cloning, Expression, and Immunogenicity of MTB12, a Novel Low-Molecular-Weight Antigen Secreted byMycobacterium tuberculosis

Infection and Immunity ◽

10.1128/iai.66.9.4208-4214.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4208-4214 ◽

Cited By ~ 29

Author(s):

John R. Webb ◽

Thomas S. Vedvick ◽

Mark R. Alderson ◽

Jeffrey A. Guderian ◽

Shyian S. Jen ◽

...

Keyword(s):

Molecular Weight ◽

Molecular Cloning ◽

Mononuclear Cells ◽

Single Copy ◽

Low Molecular Weight ◽

Peripheral Blood Mononuclear ◽

Purified Protein Derivative ◽

Culture Filtrate Proteins ◽

Processed Form

ABSTRACT Proteins secreted into the culture medium by Mycobacterium tuberculosis are thought to play an important role in the development of protective immune responses. In this report, we describe the molecular cloning of a novel, low-molecular-weight antigen (MTB12) secreted by M. tuberculosis. Sequence analysis of the MTB12 gene indicates that the protein is initially synthesized as a 16.6-kDa precursor protein containing a 48-amino-acid hydrophobic leader sequence. The mature, fully processed form of MTB12 protein found in culture filtrates has a molecular mass of 12.5 kDa. MTB12 protein constitutes a major component of the M. tuberculosis culture supernatant and appears to be at least as abundant as several other well-characterized culture filtrate proteins, including members of the 85B complex. MTB12 is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex, the BCG strain of M. bovis, and M. leprae. Recombinant MTB12 containing an N-terminal six-histidine tag was expressed in Escherichia coli and purified by affinity chromatography. Recombinant MTB12 protein elicited in vitro proliferative responses from the peripheral blood mononuclear cells of a number of purified protein derivative-positive (PPD+) human donors but not from PPD− donors.

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