Biomedical subjects

L Gigova

Publications and source records attributed to L Gigova.

6 recordsLinked to original sources

PubMed1995

Strain-specific fingerprints of Rhizobium galegae generated by PCR with arbitrary and repetitive primers.

Strain-specific genomic patterns of Rhizobium galegae were generated by PCR using both arbitrary and repetitive (BOX, ERIC and REP) primers. The identification of the strains was achieved also by RFLP analysis. However, the PCR genomic fingerprinting has significant advantages: it is not only simpler and faster, but it is also much more discriminative because it deals with the full bacterial genome and not only with parts of it as is the case with RFLP. In addition, both kinds of PCR fingerprinting (using arbitrary or repetitive primers) generated highly specific and reproducible patterns when parallel reactions with total bacterial DNA, extracted from independent liquid cultures were performed. The latter shows that AP- and rep-PCR are convenient for controlling the production and application of Rhizobium inoculants.

Base Sequence↗

PubMed1989

Expression of repetitive human calcitonin genes in Escherichia coli.

In order to stabilize recombinant human calcitonin (rhCT) against Escherichia coli proteases a series of concatemeric hCT genes with varying degrees of repetition were synthesized and expressed in E. coli under the control of a constitutive synthetic phage promoter. The series of expression vectors thus constructed was used as a model to study the effect of gene repetition on the efficiency of expression (both transcription and translation), stability of mRNA, proteolytic stability of recombinant protein, genetic stability of expression plasmids, etc. The oligomerization of the hCT gene resulted in stabilization of the mRNA increasing its half-life from 60-70 s (as in the hCT monomer, dimer, and trimer genes) to 100-120 s (for the hCT tetramer gene). This effect held true as well for the proteins coded by the corresponding repetitive hCT genes. The genetic stability (segregation and recombination) of the expression plasmids containing hCT oligomeric genes also depended on the number of hCT gene repeats. The expression plasmid containing the hCT tetramer gene segregated from one of the best producers of rhCT (E. coli LE392) up to 100% after 100 cell generations in nonselective media (free of antibiotics). One of the plasmids most sensitive to recombination events was that containing the hCT pentamer gene. The series of expression plasmids bearing hCT oligomeric genes was used for transformation of various E. coli strains in order to find the optimal host for production of rhCT. The highest yield (44-100 mg rhCT per 1 liter of bacterial culture) was obtained with the strains LE392, JM107, and DH1.

Calcitonin↗

PubMed1988

A conditional high-copy-number plasmid derivative of pBR322.

A spontaneous high-copy-number plasmid derivative of plasmid pBR322 was isolated. This plasmid bears two point mutations adjacent to the unpaired region in the stem of loop II of RNA I and expresses its high-copy-number phenotype only when the harboring cells grow on solid support (L-agar).

Base Sequence↗

PubMed1987

Chemical synthesis and expression in E. coli of a human Val8-calcitonin gene by fusion to a synthetic human interferon-gamma gene.

A gene coding for human Val8-calcitonin (Val8-hCT) was synthesized by the solid-phase phosphite approach and fused to a synthetic human immune interferon-gamma (IFN-gamma) gene. The IFN gene was previously shown to be expressed at a very high level in E. coli [(1986) Gene, in press] due to the control of a strong synthetic promoter and strong ribosome binding site. The cells harboring the fused gene produced 100-150 micrograms per l of bacterial suspension of immunoreactive calcitonin in the form of hybrid IFN-gamma-Val8-hCT protein consisting of 140 amino acids. The Val8-hCT can be released from this protein by CNBr treatment.

Amino Acid Sequence↗

PubMed1986

RNA colony hybridization method.

A method for rapid screening of specific RNA sequences in recombinant colonies by hybridization in situ is presented. The method includes two consecutive steps of lytic treatment of the nitrocellulose-filter-supported colonies (10% sodium dodecyl sulfate and 3 X SSC at 65 degrees C) and hybridization with 32P-labelled specific oligodeoxynucleotides.

Base Sequence↗

PubMed1985

Isolation of lambda phage DNA by hydroxylapatite chromatography.

A simple and rapid (1 day) method for preparation of lambda phage DNA was proposed. The method included two main steps: (a) growth and lysis of bacteria containing lambda phage and (b) purification of lambda phage DNA by hydroxylapatite chromatography. The phage DNA prepared by this method was intact and free of RNA, proteins, and bacterial DNA.

Bacteriophage lambda↗