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Biomedical subjects

C Brack

Publications and source records attributed to C Brack.

52 records · Page 3Linked to original sources
PubMed1977

Cloning of an immunoglobulin variable region gene from mouse embryo.

A 4.8-kilobase DNA fragment carrying an immunoglobulin gene coding for a mouse lambda chain variable region (Vlambda gene) was enriched about 350-fold from a total endonuclease EcoRI digest of embryonic DNA by a combination of preparative agarose gel electrophoresis of double-stranded DNA and CsCl density gradient centrifugation of R-loops formed with a purified lambda chain mRNA. DNA fragments thus enriched for the immunoglobulin gene were inserted in vitro in the middle of the genome of the vector phage lambdagt Wam 403, Eam 100, Sam 100 by use of the EcoRI cohesive ends. Transfection of CaCl2-treated Escherichia coli 803 [rk-, mk- (lacking restriction and modification systems for K-12)] with such hybrid DNA and subsequent screening of about 4000 plaques by in situ hybridization with purified 125I-labeled lambda chain mRNA led to isolation of a clone that carries a Vlambda gene (lambdagtWES-Ig 13). Electron microscopy of R-loops confirmed the presence of sequences homologous to part of the lambda chain mRNA in its 5'-end.

Animals↗
PubMed1975

Multiple steps in DNA recognition by restriction endonuclease from E. coli K.

The process of DNA recognition by the activated form of the restriction endonuclease from E. coli K involves three enzyme-DNA complexes which can be differentiated experimentally. These are: an initial complex formed at a nonspecific site; a recognition complex involving the host specificity site; and a cleavage complex dependent on the presence of ATP.

Adenosine Triphosphate↗
PubMed1975

Electron-microscopic mapping of AT-rich regions and of E. coli RNA polymerase-binding sites on the circular kinetoplast DNA of Trypanosoma cruzi.

Partial alkaline denaturation of the circular kinetoplast DNA (kDNA) of Trypanosoma cruzi has shown the existence of 4 small, well-defined AT-rich regions with an average size of about 200 base pairs. They are almost equally distributed, separated by approximately 90 degrees on the circular molecule. All minicircles, whether free or linked in networks, have the same denaturation pattern and, therefore, seem to contain the same information. The long linear molecules present in low amounts in the kDNA samples do not show the same denaturation pattern. Partial denaturation of molecules in larger associations indicates that the circular units may be linked to each other by one strand only. kDNA can be transcribed in vitro by the RNA polymerase of E. coli. RNA polymerase-kDNA complexes have been studied in the electron microscope. By spreading the DNA-protein complexes by adhesion to positively charged carbon films and dark-field observation, it was possible to show the existence of 4 specific binding sites of the E. coli RNA polymerase on the kDNA circles. Comparing the position of the polymerase-binding sites and the AT-rich melted zones, it is suggested that a correlation exists between the two. As had been shown in earlier work, the replication of circular kDNA can be blocked by treating the trypanosomes with the trypanocidal drug Berenil. The comparison of the relative position of the Berenil-blocked replication forks with the position of the 4 denaturation loops shows that the DNA replication is stopped at these AT-rich regions. Since there is evidence that Berenil binds preferentially to AT-rich DNA and seems to be involved in inhibition of DNA replication, the following hypothetical model can be proposed. The replication of the circular kDNA molecules is discontinuous and involves the synthesis of RNA primers; when Berenil is bound to the AT-rich regions, synthesis of new RNA primers is inhibited and replication is blocked at these points, leading to the accumulation of replicating intermediates with defined branch lengths.

Animals↗
PubMed1972

Replicating, convalently closed, circular DNA from kinetoplasts of Trypanosoma cruzi.

When Trypanosoma cruzi are treated with Berenil, a trypanocide, their kinetoplast DNA contains an increased proportion of double-branched circular molecules. These replicating molecules have closed-circular template strands; their decrease in density when complexed by ethidium bromide in a cesium chloride gradient is proportional to the length of the replicated segments. Replication seems to be blocked at specific points, which are equidistantly spaced along the circular kinetoplast DNA molecules. Analysis of about 800 replicating forms showed that the lengths of the replicated branches are not distributed at random, but into several populations, which correspond to multiples of 15% of the total contour length of 0.5 mum. This distribution evokes a discontinuous replication process. The problem of whether kinetoplast DNA is synthesized by successive replication units, or whether and how Berenil might induce specific blocking of DNA replication, is discussed.

Acetates↗
PubMedDate not supplied

DNA bending induced by specific interaction of decamer binding proteins with immunoglobulin gene control sequences.

In order to investigate the properties of specific DNA-binding proteins involved in tissue-specific regulation of immunoglobulin genes, we have analyzed the interaction of nuclear proteins from mouse B-cell hybridomas with promoter and enhancer sequences of a mouse immunoglobulin heavy chain gene. Visualization of specific complexes has shown that protein binding induces a sharp bend at the position of the conserved decamer sequence. After fractionation of nuclear extracts, several sequence-specific DNA binding proteins could be distinguished by UV crosslinking to radioactive synthetic oligonucleotides. Decamer binding factor I (DBF-I) a protein of 100-105 kDa and DBF-II, a family of proteins of 25-35 kDa were purified on specific DNA-affinity columns. Both proteins bend the DNA at the dc sequence as shown by electron microscopy and by gel retardation. These data suggest that one possible function of sequence-specific regulatory proteins may be to locally change the DNA topology, thereby facilitating the interaction of additional transcription factors with the primary complex.

Animals↗