Isolation and characterization of human actin genes cloned in phage lambda vectors

Using Drosophila and chicken actin probes, we have selected 14 human actin lambda recombinants from a genomic library. We present a restriction maps indicating the positions of the sequences homologous to actin and to an Alu probe. Restriction mapping has revealed that nine out of ten of these clones are distinct, indicating that actin is a multigene family. Hybrid elution of HeLa cell mRNA from filters containing the recombinant DNA, followed by in vitro translation and immunoprecipitation, as well as one- or two-dimensional protein analysis, shows that these recombinants code for actin. Hybridization back to human DNA digested with restriction enzymes shows that the EcoRI fragments of at least one of the lambda recombinants (lambda HA-5) result in similar-sized human DNA fragments in the intact genome. In nuclei, a 4.5-kb mRNA precursor to the cytoplasmic 1.9-kb mRNA can be detected by hybridization with genomic or cDNA probes, indicating the presence of additional sequences and RNA processing.     

CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects.

The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.     

Use of DNA-protein interaction to isolate specific genomic DNA sequences.

We describe a simple and rapid method for the isolation of specific genomic DNA sequences recognized by DNA-binding proteins. This procedure consists of four steps: (1) restriction enzyme digestion and size fractionation of genomic DNA; (2) DNA--protein binding using the gel mobility-shift assay; (3) ligation of isolated DNA fragments followed by transformation of Escherichia coli; and (4) screening of recombinant clones for inserts containing specific DNA--protein binding sequences. We have used this protocol to isolate human DNA sequences, 100-200 bp in size, that are recognized by both partially purified and affinity purified proteins. Unlike other procedures designed to identify genomic target sequences, the method described does not require polymerase chain reaction or successive immunoprecipitations.     

DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.     

On the influence of thymidine analogues on the activity of phage fd promoters in vitro

RF I DNA of phage fd containing 5-bromo-deoxyuridine (br5Ud) or deoxyuridine (Ud) instead of deoxythymidine (Td) inthe codogenic strand was synthesized in vitro. The modified genomes could be cleaved by restriction endonuclease Hpa II. Although the recognition site of Hpa II is CCGG, the cleavage rate was significantly reduced with Ud-containing DNA. Both base substitutions altered the mobilities of several DNA fragments under the conditions of polyacrylamide gel electrophoresis. The fragments containing binding sites for RNA polymerase were assayed for the rates of stable complex formation. The substitution of Td for both, Ud and br5Ud, strongly influenced this parameter. Thus the methyl group of Td has to be regarded as one of the sites in DNA which determine the rate of stable RNA polymerase binding and thereby possibly mediate promoter activity in vitro (24,25,26). In most cases the rate of complex formation was decreased by Ud, but increased by br5Ud.     

Construction of promoter-probe plasmid vector

A new plasmid vector, designated pBRS188 has been constructed for cloning of promoter-containing DNA fragments. This plasmid is a derivative of the E. coli drug-resistance plasmid pBR322 in which a small region (13 base pairs long) within the Tc promoter is eliminated. As a result of the alteration pBRS188 has lost the ability to confer Tc resistance to the host strain. Cloning of foreign DNA fragments, carrying promoters for E. coli RNA polymerase, into the unique EcoRI site of pBRS188 allows to isolate the recombinant TcR transformants. Our construction required the use of new techniques, involving partial hydrolysis of DNA fragments by E. coli DNA polymerase I in the presence of one deoxyribonucleosidetriphosphate and by nuclease S1. An important feature of this method is the ability to regenerate restriction endonuclease recognition sites at junctions of DNA fragments.     

Rolling circle amplification of genomic templates for inverse PCR (RCA–GIP): a method for 5′- and 3′-genome walking without anchoring

We have devised an improved method of genome walking, named rolling circle amplification of genomic templates for Inverse PCR (RCA–GIP). The method is based on the generation of circular genomic DNA fragments, followed by rolling circle amplification of the circular genomic DNA using ϕ29 DNA polymerase without need for attachment of anchor sequences. In this way from the circular genomic DNA fragments, after RCA amplification, a large amount of linear concatemers is generated suitable for Inverse PCR template that can be amplified, sequenced or cloned allowing the isolation of the 3′- and 5′- of unknown ends of genomic sequences. To prove the concept of the proposed methodology, we used this procedure to isolate the promoter regions from different species. Herein as an example we present the isolation of four promoter regions from Crocus sativus, a crop cultivated for saffron production.