Hind III genomic polymorphism of the C3b receptor (CR1) in patients with SLE: low erythrocyte CR1 expression is an acquired phenomenon.

Expression of the human erythrocyte C3b receptor (CR1-CD35) and its Hind III RFLP was studied in a group of 37 patients with SLE, 15 consanguineous relatives of the patients and 48 healthy normal subjects. The CR1 number on erythrocytes was quantitated by ELISA using a mAb to CR1. Serum levels of complement proteins (C3, C4, C3d) and circulating immune complexes (CIC) were estimated simultaneously in controls and relatives. The patients were followed up during the course of the treatment. The CR1/erythrocyte (CR1/E) in patients were found to be significantly low in comparison to controls. The gene frequencies for the alleles H and L (7.4 and 6.9 kb Hind III restriction fragments) in the patients were 0.75 and 0.25, respectively, which did not differ significantly from the controls (0.77 and 0.23 in normal subjects and 0.79 and 0.21 in consanguineous relatives of the patients). However, patients expressed fewer CR1/E within each genotype than their relatives and healthy subjects. CR/E was found to be stable in consecutive samples in controls. In patients, the numbers varied between low and high during the course of the treatment. The variation in the numbers was significantly correlated with C3d and CIC as well as with the severity of the disease. Our results suggest that low levels of CR1 on erythrocytes in SLE patients are required during the course of the disease and that the 6.9 kb restriction fragment does not play a role in causing susceptibility to the disease.

Molecular definition of unique species status of Mycobacterium w; a candidate leprosy vaccine strain.

Mycobacterium w, a candidate leprosy vaccine strain, is an atypical cultivable mycobacterium. Based on its growth and metabolic properties, M. w was listed in Runyon Group IV, along with other rapid growers such as M. fortuitum, M. smegmatis, M. chelonae and M. vaccae. However, M. w was not fully identical to any one of these. In the present study, a molecular biology approach was used to define the species identity of M. w in a manner that allows reliable comparison to be made with over 30 known mycobacterial species. A 383-bp region, present at the amino terminus of the conserved mycobacterial 65-kDa gene, has been polymerase chain reaction (PCR) amplified in M. w and DNA sequence was determined. A comparison of the M. w DNA sequence with those of M. tuberculosis, M. avium, M. paratuberculosis and M. fortuitum revealed a species-specific polymorphism, i.e., the presence of nucleotide substitutions unique to M. w. In an alternate approach, a 441-bp region, also a part of the 65-kDa gene, has been PCR amplified in M. w and a Hae III restriction pattern was generated.(ABSTRACT TRUNCATED AT 250 WORDS)

C3b receptor (CR1) genomic polymorphism in rheumatoid arthritis. Low receptor levels on erythrocytes are an acquired phenomenon.

The number of complement receptor 1 (CR1, CD35) molecules on erythrocytes is genetically determined by two codominant alleles. The numerical expression of CR1 on erythrocytes correlates with a HindIII-RFLP or CR1 gene using CR1-1, a complementary DNA probe. We have found low CR1 on erythrocytes in patients with rheumatoid arthritis (RA) in an Indian population. Low levels in RA patients may be acquired or genetically determined. Fifty-two patients with RA, 48 nonrelated healthy subjects and 19 consanguineous relatives of patients were genotyped. CR1 numbers on erythrocytes were quantitated by the enzyme-linked immunosorbent assay using monoclonal anti-CR1 antibody. Normal subjects and patients were followed up for a period of 6 months to evaluate the stability of their CR1 expression. The gene frequency for allele H and L (7.4- and 6.9-kb HindIII restriction fragment, respectively), which correlated with high and low expression of CR1 on erythrocytes was 0.77 and 0.23 in the normal controls. Gene frequency in RA patients was 0.78 and 0.22 for H and L allele, which did not differ significantly from either controls or relatives (0.80 and 0.20 for H and L allele, respectively). However, RA patients expressed fewer CR1 on erythrocytes within each genotype than their relatives and controls. CR1 on erythrocytes were found to be stable in consecutive samples in controls. In RA patients, the number varied between low and high during the course of the disease. The variation in number was significantly correlated (p < 0.05, r = -0.85 to -0.98) with disease activity as monitored by erythrocyte sedimentation rate. Our results suggest that low levels of CR1 on erythrocytes in patients with RA are not inherited, rather they are acquired during the course of the disease.

Molecular cloning and characterization of contiguously located repetitive and single copy DNA sequences of Mycobacterium tuberculosis: development of PCR-based diagnostic assay.

Screening of a lambda gt11 genomic library has been used as an approach for molecular cloning of the Mycobacterium tuberculosis repetitive DNA shown to be present on a previously described 5.6-kb Alu I genomic fragment. The recombinant clone R18.8.2, which strongly hybridized with the radiolabeled 5.7-kb Alu fragment, carried two Eco RI inserts of 2 kb and 1.4 kb in size. Southern hybridization of each of these fragments to restriction endonuclease-cleaved M. tuberculosis DNA clearly demonstrated the 2 kb to contain the repetitive DNA sequence, while the 1.4 kb is represented in a single copy. When replica plaque lifts from the genomic library were probed, the 5.6-kb genomic fragment and the cloned 2-kb repetitive insert hybridized to an identical number of plaques, indicating the similarity and the high copy number of the repeating unit shared by the two fragments. Restriction mapping and Southern hybridization patterns indicated that the 2-kb repetitive and the 1.4-kb single-copy DNA sequences originated from a contiguous piece of genomic DNA. Both fragments were found to be unique to members of the M. tuberculosis complex, except that the 2-kb insert exhibited a weak hybridization with M. kansasii DNA. Finally, a 169-bp region from one end of the single-copy sequence has been amplified by polymerase chain reaction (PCR) in a manner specific to members of the M. tuberculosis complex. The sensitivity of the PCR was such that as few as 9-10 bacilli could be detected.

Cloned single copy DNA sequences of Mycobacterium tuberculosis as DNA probes.

A recombinant genomic clone was isolated from a lambda gt 11 library of M. tuberculosis on the basis of lack of hybridization with M. avium and M. kansasi. The specificity and sensitivity of M. tb DNA probes, 2.5 and 2.3 kb in size, were assessed by Southern blot and dot blot hybridization. These did not cross hybridize to DNA of mycobacteria other than members of M. tb complex, nor with DNA of non mycobacterial origin. Sensitivity was determined to be 200 pg which is equivalent to 10(4) bacilli. Genomic Southern hybridization indicated single copy nature of the probes.

Repetitive DNA sequence of Mycobacterium tuberculosis: analysis of differential hybridization pattern with other mycobacteria.

In order to generate specific DNA probes for Mycobacterium tuberculosis, restriction fragment length analysis was carried out with some of the mycobacterial species that fall within the tuberculosis complex. The presence of specific bands of 5.6 kb and 4.8 kb was revealed in the AluI DNA digest of M. tuberculosis. The hybridization profile of the 5.6-kb AluI DNA sequence, as judged by the Southern blot and dot blot hybridization experiments, revealed the presence of this sequence in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG as multiple copy and M. kansasii as single copy but this sequence was not present in M. avium, M. smegmatis, or M. vaccae genomes. Genomic clones corresponding to the 5.6-kb AluI fragment from M. tuberculosis H37Rv library made in the lambda gt11 expression vector were isolated.

Construction of genomic libraries of mycobacterial origin: identification of recombinants encoding mycobacterial-specific proteins.

A complete genomic library from Mycobacterium vaccae (2785 recombinants) and a partial genomic library of M. leprae and BCG (300 and 1750 clones, respectively) were constructed in the plasmid pBR322. Bam HI was selected as the restriction endonuclease for obtaining DNA cleavage products. Evidence was obtained for limited expression of the cloned mycobacterial DNA inserts in Escherichia coli. A recombinant has been identified which codes for antigen immunoreactive with rabbit anti-M. leprae antibody but not with anti-H37Rv antibody.

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.

Transport in bacteriophage P22-infected Salmonella typhimurium.

There was rapid efflux of L-leucine, L-phenylalanine, and alpha-methyl-D-glucoside after infection of Salmonella typhimurium with the clear plaque mutant C1 of phage P22. The efflux was similar to that observed with cyanide or arsenate treatment except that there was partial recovery in the case of phage infection and almost complete recovery under the condition of lysogeny. There was no efflux after infection with the temperature-sensitive mutant ts16C1 at nonpermissive temperature. Superinfection of superinfection exclusion negative lysogen (sie A minus sie B minus) with C1 led to efflux, whereas the efflux was much less on superinfection of sie A+ Sie B+ lysogen. These results indicate that an effective injection process is enough to cause depression in the cellular transport processes.

Effect of m3 gene on the development of phage P22.

Mutation of the gene m3 of phage P22 causes permanent depression of macromolecular synthesis in the infected host and thus inhibits phage development as indicated by burst size and lysozyme production. The permanent depression of macromolecular synthesis is most probably due to blockage of the transport process. The m3 allele is dominant over m+. m3 allows some transcription of phage genes (however, the difference between early and late function is not clear). The inhibitory effect of m3 on DNA synthesis may be indirect.