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The crossing over in five murine I-region recombinants (Is/Ik) was studied by restriction fragment length polymorphism analysis after Southern blot hybridization by using I-region-specific probes. These recombinants included three recently developed strains, B10.ASR1, B10.ASR11, and B10.ASR12; and two strains B10.S(9R) and B10.HTT derived earlier. Although these recombinants were reciprocal in haplotype orientation to the three recombinants we reported recently, these too crossed over within the same 7 kb stretch of DNA in the E beta gene. This 7 kb stretch of DNA included the 3' half of the first intron, the beta 1 exon, the second intron, and the beta 2 exon. A comparison of the cDNA sequences of the two parental E beta alleles revealed that although the beta 2 exons were identical, there were several nucleotide differences between the two beta 1 exons. This allowed us to determine the parental origin of the beta 1 exon in the recombinants at the level of transcription by using S1 nuclease mapping. Thus we were able to show that in each case the 3' portion of the first intron and the beta 1 exon were upstream from the site of crossover. All eight recombinants involving the k and s haplotypes now can be mapped within a 4.5 kb stretch of DNA, which includes only the beta 1-beta 2 intron and the beta 2 exon of the E beta gene. These findings imply that the I-E molecules expressed in these recombinants will probably have conserved sequences and therefore will exhibit identical I-E-restricted immune responses, despite the fact that crossing over could have occurred at different sites within the beta 1-beta 2 intron.

Copolymers of poly/vinyl alcohol-acrylic acid/ with various content of sulphate and carboxyl groups have been synthetized and tested for their in vitro effect on blood coagulation. The results indicate that the sulphated copolymers display an inhibitory effect but there is a requirement in the charged groups of about 20% in the molecule to possess effective anticoagulation. The biochemical mechanism of their actions is complex, i.e. the inhibition of blood clotting is a consequence of both the accelerated inactivation rate of thrombin by antithrombin-III and a direct inhibitory effect on the thrombin-fibrinogen reaction. Moreover, additional effects may occur on other blood coagulation enzymes than thrombin, depending on the chemical composition of the copolymers.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.

Sets of genes under a common regulatory control in a given cell type are often differentially transcribed. The possibility that this differential transcription can be modulated by the number or strength of cis-acting regulatory sequences associated with a given gene was tested by using the glucocorticoid-responsive enhancer element associated with the mouse mammary tumor virus promoter. Results indicate that differential levels of hormone-inducible gene expression can be modulated in an additive way by the number of glucocorticoid-responsive enhancers associated with this promoter. Realization of these effects shows little preference for position of the additional elements with respect to the promoter. When sequences that bind the glucocorticoid receptor in vitro with somewhat lower affinity than the enhancer were tested, these additive effects were not detected. The results support that differential transcription of genes subject to a common regulatory control can be mediated, at least in part, by the number or strength of their associated cis-acting regulatory sequences.

Enzymeimmunoassay (EIA) with monoclonal antibody against human estrogen receptor (ER) from MCF-7 breast cancer cells and radioimmunoassay (RIA) with monoclonal antibody against ER D-5 antigen from human myometrium were applied to human breast tumor, and the results were compared to those of steroid binding assay using the dextran coated charcoal (DCC) method. The rates of coincidence of positivity and negativity of ER between the DCC method and EIA or RIA in 30 human breast tumors were 96.7% (29/30) and 86.7% (26/30), respectively. A highly significant positive correlation was observed between ER values obtained by the DCC method and those by EIA (r = 0.87, P less than 0.001). On the other hand, the correlation between ER values obtained by the DCC method and those by RIA was less significant (r = 0.41, P less than 0.05). The discrepancy between these ER values may be due to the difference of the monoclonal antibody used in each new assay. The above results indicate that EIA and RIA with monoclonal antibody for human ER are useful for clinical use, although the reason for the partial discrepancy of the data between the DCC method and RIA remains to be elucidated.

Monoclonal antibodies against estrogen receptor (ER) were used for determination of ER status immunocytochemically in histologic specimens from 192 primary breast carcinomas. All tumors were also assayed biochemically for ER with the dextran-coated charcoal method (DCC). The comparison of biochemically and immunocytochemically determined ER status showed concordant results in 80% (P less than 0.0001). In only 2 cases (1%) with low ER levels (less than 20 fmol/mg protein) immunocytochemistry failed to detect ER. ER positivity determined with a semiquantified approach based on intensity and heterogeneity of immunocytochemical staining correlated significantly with biochemically determined ER levels (P = 0.0001). In a series of fine-needle aspirates of 34 breast carcinomas sufficient cell material was available for ER immunocytochemistry (ER-ICA). Overall, the results of ER-ICA in fine-needle aspirates were concordant with ER-ICA in histologic specimens in 88% of the samples. In a few cases with weak positivity of ER-ICA in histologic specimens, ER-ICA was negative in fine-needle aspirates. In no case was there a false-positive immunocytochemical ER determination in a tumor aspirate. Thus, ER-ICA seems to be a reliable assay which can be performed in histologic and cytologic specimens.

No abstract available.