Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP.

We provide fast, simple, one-step procedures for sequence-specific detection of nucleic acids in situ. Tandem repeat sequences in DNA are stained within 30 min, and mRNA is stained within 2 h. The procedures are based on the incorporation of the newly available fluorescein-labeled dUTP into DNA synthesized in situ by primed in situ labeling, with denatured fragments of cloned DNA or oligonucleotides as primers. The extreme speed and simplicity of the reaction make it attractive for automatization in routine laboratory procedures and opens up new diagnostic possibilities.     

Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase delta.

The cDNA of human DNA polymerase delta was cloned. The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa. Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb. Monoclonal and polyclonal antibodies to the C-terminal 20 residues specifically immunoblotted the human pol delta catalytic polypeptide. A multiple sequence alignment was constructed. This showed that human pol delta is closely related to yeast pol delta and the herpes virus DNA polymerases. The levels of pol delta message were found to be induced concomitantly with DNA pol delta activity and DNA synthesis in serum restimulated proliferating IMR90 cultured cells. The human pol delta gene was localized to chromosome 19 by Southern blotting of EcoRI digested DNA from a panel of rodent/human cell hybrids.     

Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). Methodological aspects and application to routine diagnostic material.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) has been suggested as a new approach for determining proliferative activity in paraffin-embedded tissue. In a prospective study PCNA immunostaining was performed in 284 colorectal biopsies using monoclonal antibodies 19F4 (Ogata et al. 1987) and PC10 (Waseem and Lane 1990) and compared with the Ki67 method. From each site three biopsies were taken and a variety of fixation regimens for frozen and paraffin-embedded samples tested. For frozen biopsies methanol fixation at -20 degrees C proved best. In paraffin sections PCNA could be detected after methacarn fixation as well as after controled fixation at 4 degrees C in 4% paraformaldehyde for 1 h and in most biopsies routinely fixed with 10% formalin. However, the latter fixation regimens revealed additional PCNA-positive cells in the normal superficial colonic mucosal epithelium. Although the percentage of cells positive for PCNA was generally lower than for Ki67, the rates correlated in a highly significant fashion, both in frozen methanol-fixed biopsies, and in paraformaldehyde-fixed paraffin-embedded samples. PCNA immunohistochemistry revealed a similar proliferative activity in different parts of the large bowel. A higher proliferative activity was found in inflamed mucosa, adenomas, carcinomas and even in normal mucosa from patients with colorectal neoplasms. In routinely fixed biopsies, the monoclonal antibody PC10 was superior to 19F4 because of considerably less background staining. However, in the routine material only a rough estimate of the proliferative activity was possible by PCNA immunohistochemistry using these antibodies, because unpredictable numbers of non-S-phase cells were also stained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in anti-oncogene p53 expression in human monocytes and lymphocytes in vitro]

The p53 gene has been associated with malignant transformation as well as "anti-oncogene" activity. In the present report expression of p53 in resting and activated human blood monocytes and lymphocytes is analyzed. It is found that human monocytes freshly isolated by continuous percoll gradient centrifugation contained detectable level of p53 mRNA. Stimulation of monocytes by potent activation inducer Staphylococcus Aureus Cowan I for 3-5 hr caused disappearance of r53 mRNA. In contrast, induction of high level of TNF-alpha mRNA was detected. Addition of cycloheximide had no effect on p53 mRNA content in stimulated monocytes, and caused disappearance of mRNA in resting cells. In lymphocytes cultures p53 mRNA was absent in freshly isolated cells and in resting lymphocytes cultured for 20 hr. Activation of lymphocytes by lectin caused accumulation of p53 mRNA. We suggest that r53 gene regulation and functions might be different in human monocytes and lymphocytes.     

Use of commercial highly sensitive tests for detecting HbsAg in healthy persons]

The study has revealed the possibility of the contamination of serum with HBsAg under laboratory conditions during its treatment and preparation for analysis, which may be the cause of false positive results of HBsAg detection by the enzyme immunoassay (EIA). Excluding the factors of contamination, the authors demonstrate the efficacy of commercial systems, such as the passive hemagglutination test system "Gorky" and the EIA systems "Gorky" and "DIAplus", for the detection of HBsAg in the blood of donors in Moscow and Tashkent.     

The phages of halophilic vibrios and their use]

The range of the lytic activity of 46 phages of parahemolytic vibrios isolated from lysogenic strains, sea water samples, crabs and mussels has been studied. The phages are represented by virions belonging to morphological groups II, IV, V according to the phage classification currently used in Russia and to different serological groups. No relationship between the sensitivity of vibrio strains to the phages under study and the specificity of serotypes O and K has been established. The preparation of diagnostic phage [see text] suitable for the identification of 82% of strains of parahemolytic vibrios has been proposed.     

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.