Gene analysis techniques最新文献

A procedure that allows one to directly detect baculovirus recombinants that express foreign proteins of interest in situ using antibody probes is described. Nitrocellulose replicas of recombinant plaques are probed with specific antibodies, and the antibody antigen complexes are detected using alkaline phosphatase conjugated secondary antibody. This procedure was used to isolate a baculovirus recombinant that expresses Vmw65, the Herpes simplex virus transacting factor that is involved in the induction of viral immediate early gene transcription. Immunoscreening directly on plaque lifts is rapid and reliable and facilitates the testing of different transfer vectors for optimization of foreign gene expression.

A 41-nucleotide-long duplex DNA, which contains the translation termination codon TAA in six reading frames and lactose operator sequence of Escherichiacoli, has been synthesized. This fragment may be useful not only for producing a truncated protein encoded in a plasmid, but also for the identification of the precise coding region and translation direction of a bacterial gene in the cloned chromosomal segment. The synthetic fragment was inserted into ß-lactamase structural gene in pBR322 in order to test the in vivo activity. The plasmid produced mutant ß-lactamase reduced in size, as expected from the insertion site, and rendered the host bacterium constitutive for ß-galactosidase. Thus, termination codons and lactose operator in synthetic nucleotide appear to be functional in vivo.

Our previously reported data from DNA sequence studies of the c-myc locus show that the human c-myc exon 1 has an open reaading frame capable of encoding a protein of 188 amino acid residues. To confirm the presence of the open reading frame, we constructed a recombinant vector (pMCP60) that contains a segment of the λ cII translational initiation region, a portion of the N-terminus of the v-mos gene, and 639 base pairs of the first exon of the human c-myc gene. pMCP60 expresses a 38 kilodalton tripartate protein (cII-mos-myc), which was purifed by high-pressure liquid chromatography. The presence of myc exon 1 sequences in the cII-mos-myc fusion protein was confirmed by partial amino acid sequence analysis. These experiments further establish that the first exon of the human c-myc gene contains an open reading frame capable of expressing a protein in Escherichia coli.

The study of gene expression in cells and tissues often begins with phenol-chloroform extraction of the biologic material of interest for the isolation of intact mRNA. In most cases, the proteins denatured by phenol-chloroform are discarded. However, we found that the proteins recovered from phenol-chloroform extractions maintain their antigenicity. Therefore a method was developed for recovering the proteins from phenol-chloroform-denatured extracts that could be saved in lyophilized form until immunologic analysis. In this way, the RNA and the protein analysis can utilize exactly the same sample, and the biologic material can be saved. This is important because often these materials are available only in limited quantities. The method has been used to examine the sea urchin ets-related antigen and sea urchin ets-2 mRNA.

A general and sensitive detection method of target DNA is described. The system is based on an oligonucleotide probe labeled to high specific activity. This involves a novel oligonucleotide design incorporating at the 3′ end a hairpin structure, allowing extension by polymerase reaction.

Assay of endonuclease activity, as performed in most laboratories, depends upon change in form of small, defined substrate molecules, with a secondary computation required to obtain a determination of enzyme activity. We now explore the assumptions inherent in these computations and provide a series of equations that permit more accurate determinations of enzyme activity from assays of this type. These equations allow information to be obtained not only from substrate fractions left uncleaved by the endonuclease, upon which conventional systems rely, but also from products cleaved by the enzyme. Some information yielded by these equations is unobtainable using conventional methods of analysis.

We have designed a simple procedure for the construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent cloning vector. An oligonucleotide, its 5′ end starting with a heteropolymeric sequence encoding the rare restriction sites for NotI and SfiI, followed by 50 dT residues, is used to prime first-strand synthesis on size-selected mRNA. After second-strand synthesis and EcoRI linker addition, the cDNA is double digested with EcoRI and NotI, or with EcoRI and SfiI, to generate DNA fragments with asymmetric ends that can be directionally cloned. The cDNA fragments are enriched for “full length” by size selection and ligated into a phage lambda vector containing the T3 and T7 RNA polymerase promotors. These cDNA libraries can directly be used for in vitro synthesis of sense or antisense RNA.

Avian myeloblastosis virus reverse transcriptase (AMV RT) is routinely used in the sequence analysis of RNA and DNA templates. We review the various methods for dealing with secondary structures that would otherwise result in premature termination or sequence compression. Based on our experience in sequencing the 11-kb single-stranded RNA genome of lymphocytic choriomeningitis virus, we have found that raising the reaction temperature above 47°C is the simplest way to overcome template secondary structure, and the use of 98% formamide gels is the simplest way to overcome product secondary structure.

Plasmids pWQX001 and pWQX005, constructed from pGEM-4 with an insert of 6.5 kb, were unidirectionally digested with exonuclease III and exonuclease VII. The DNA digests were ligated and used to transform competent cells of Escherichia coli DH5-alpha. The size of the deletion plasmid carried by each transformant was estimated through agarose gel electrophoresis of crude lysates without any purification of the plasmid DNA. Colonies carrying plasmid DNAs with different deletions of the insert were grown and their DNAs were purified through a miniprocedure. The size of each purified plasmid DNA was determined accurately after linearization of the plasmid with an appropriate restriction endonuclease. The remainder of the DNA preparation was sufficiently pure to be sequenced using Sanger's dideoxynucleotide chain termination method. An easy, quick procedure is described for the preliminary selection of templates for DNA sequencing after construction of deletion clones of recombinant plasmid DNA using exonuclease III and exonuclease VII. This procedure permits a rapid screening of large numbers of colonies and selection of those carrying plasmid DNAs with inserts of the desired sizes for sequencing. This procedure does not require purification of the deletion plasmid DNA.

A series of plasmids were constructed to generate RNA complementary to the β-galactosidase messenger RNA under control of the phage lambda P_L promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda P_L promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5′ and/or the 3′ region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of β-galactosidase synthesis occured when a functional ribosome binding site was present near the 5′ end of the anti-mRNA and the anti-mRNA synthesized was complementary to the region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5′-coding sequence. anti-mRNAs producing maximal inhibition of β-galactosidase synthesis exhibited an anti-lacZ mRNA: normal lacZ mRNA ration of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA: normal lacZ mRNA ratios. A functional ribosome binding site at the 5′-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorppration of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of β-galactosidase synthesis.