The 65-kilodalton antigen of Mycobacterium tuberculosis.

The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.     

Two integrated partial repeats of simian virus 40 together code for a super-T antigen.

We determined that the coding sequence for a 100-kilodalton super-T antigen found in Simian virus 40 mouse transformants spanned two separate partial repeats of the viral genome. The downstream repeat contained a complete Simian virus 40 large-T-antigen gene, whereas the upstream repeat was a truncated copy of the same gene. When the repeats were separated by subcloning, the capacity to code for the super-T antigen was lost. A small insertion or deletion in the origin-control region which preceded the second repeat could also destroy the ability to code for the 100-kilodalton protein. Our data suggest that differential splicing between parts of two gene copies was responsible for the additional molecular weight of this super-T antigen.     

Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii.

DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.     

Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D3 modulated protein from embryonic chick intestine

An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport.     

Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen.

The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases.     

Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus.

Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera. The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169. A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11. Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment. The gene was transcribed into a late 1.3-kilobase RNA. The nucleotide sequence of the coding region was determined. Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase. In Western blots these proteins were recognized by human sera. Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots. In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated.     

Escherichia coli F41 adhesin: genetic organization, nucleotide sequence, and homology with the K88 determinant.

The genetic organization of the polypeptides required for the biosynthesis of the F41 adhesin of enterotoxigenic Escherichia coli strains was investigated. Maxicell analysis demonstrated that a recombinant plasmid which mediated mannose-resistant hemagglutination and F41 antigen production encoded four polypeptides of 29, 30, 32, and 86 kilodaltons. The 29-kilodalton protein was identified as the F41 antigen, and the nucleotide sequence of the gene was determined. Extensive homology was observed between the region encoding the putative signal sequences of the F41 and K88 antigens and in the region immediately upstream of the antigen genes. The nucleotide sequence homology between F41 and K88 determinants was further investigated by Southern blot hybridization. A K88 probe hybridized at high stringency to all fragments shown to be essential for F41 production except for fragments internal to the F41 antigen gene.     

Characterization of the promoter, signal sequence, and amino terminus of a secreted beta-galactosidase from "Streptomyces lividans"

The gene for a secreted 130-kilodalton beta-galactosidase from "Streptomyces lividans" has been cloned, its promoter, signal sequence, and amino terminal region have been localized, and their nucleotide sequence has been determined. The signal sequence extends over 56 amino acids and shows the characteristic-features of signal sequences, including a hydrophilic amino terminus followed by a hydrophobic core near the signal cleavage site. The secretion of beta-galactosidase depends on the presence of the signal sequence. beta-Galactosidase is the major protein in culture supernatants and extracts of strains expressing the cloned beta-galactosidase gene and represents a valuable tool in the study of protein secretion in Streptomyces spp.     

A class of soybean low molecular weight heat shock proteins : immunological study and quantitation

Two major polypeptides of the 15- to 18-kilodalton class of soybean (Glycine max) heat shock proteins (HSPs), obtained from an HSP-enriched (NH₄)₂SO₄ fraction separated by two-dimensional polyacrylamide gel electrophoresis, were used individually as antigens to prepare antibodies. Each of these antibody preparations reacted with its antigen and cross-reacted with 12 other 15- to 18-kilodalton HSPs. With these antibodies, the accumulation of the 15- to 18-kilodalton HSPs under various heat shock (HS) conditions was quantified. The 15- to 18-kilodalton HSPs began to be detectable at 35° C, and after 4 hours at 40° C they had accumulated to a maximum level of 1.54 micrograms per 100 micrograms of total protein in soybean seedlings and remained almost unchanged up to 24 hours after HS. Accumulation of the HSPs was reduced at temperatures higher than 40° C. At 42.5° C the HSPs were reduced to 1.02 micrograms per 100 micrograms, and at 45° C they were hardly detectable. A brief HS at 45° C (10 minutes), followed by incubation at 28° C, which also induced HSP synthesis, resulted in synthesis of this class of HSPs at levels up to 1.06 micrograms per 100 micrograms of total protein. Taking into consideration the previous data concerning the acquisition of thermotolerance in soybean seedlings, our estimation indicates that the accumulation of the 15- to 18-kilodalton HSPs to 0.76 to 0.98% of total protein correlated well with the establishment of thermotolerance. Of course, other HSPs, in addition to this group of proteins, may be required for the development of thermotolerance.     

Polyclonal Antisera to the Individual Neurofilament Triplet Protdins: A Characterization Using ELISA and Immunoblotting

In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68- and 150-kilodalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200-kilodalton neurofilament polypeptide cross-reacted to some extent with the 150-kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme-linked immunosorbent assay (ELISA), and the cross-reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results in the immunoblots, the antiserum against the 200-kilodalton neurofilament polypeptide was subunit-specific, as was the 150-kilodalton antiserum. The 68-kilodalton antiserum displayed a minute cross-reactivity against bovine 150- and 200-kilodalton neurofilaments, but it cross-reacted somewhat more with the rat 150- and 200-kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.     

Isolation and characterization of a cDNA encoding a synaptonemal complex protein.

A gene encoding a 65-kilodalton antigen of the rat synaptonemal complex, SC65, has been cloned by screening rat testis lambda gt11 and lambda ZAPII cDNA expression libraries using polyclonal antibodies against rat synaptonemal complex proteins. The longest open reading frame, initiating at an ATG codon in the cDNA, encodes a protein of 431 amino acids, with a relative molecular mass of 50,000. Immunological analysis locates the SC65 gene product on the synaptonemal complex between the pairing faces of the parallel aligned cores of homologous chromosomes in spermatocytes. Of the rat tissues examined, the SC65 gene is transcribed in testis, brain, and heart at similar levels, and in the liver at a much lower level. The DNA sequence extending about 80 base pairs downstream of the translation termination codon has 93% similarity to the identifier sequence present in the rat genome in 1 x 10(5)-1.5 x 10(5) copies and in cDNA clones of precursors of brain-specific mRNAs. The amino acid sequence encoded by the SC65 gene contains an acidic region in the C-terminal domain of the protein, potential glycosylation sites, and at least one possible phosphorylation site. The protein shows no overall similarity to proteins of known function, nor is there similarity to protein sequences present in GenBank or EMBL data bases.     

Molecular cloning of a novel human leukemia-associated gene. Evidence of conservation in animal species

We have recently described an 18-kilodalton polypeptide (p18) that is present in much greater abundance in acute leukemic blast cells (myeloid and lymphoid) than in resting or proliferating nonleukemic lymphoid cells or chronic lymphoid and myeloid leukemic cells. In this report we describe the cloning of two different sized full-length cDNAs that code for p18. The two cDNAs differ in their 3'-noncoding regions as a result of alternative polyadenylation. Analysis of the complete nucleotide sequence and the corresponding amino acid sequence did not reveal significant homology to any previously described sequences. We show evidence that this gene is highly conserved in several animal species and low stringency hybridization studies suggest that the p18 gene may be a member of a family of partially homologous genes in the human genome.