Biomedical subjects

R Maldonado-Rodriguez

Publications and source records attributed to R Maldonado-Rodriguez.

4 recordsLinked to original sources

PubMed1991

In vitro mutagenesis in the lacI gene of Escherichia coli: fate of 3'-terminal mispairs versus internal base mispairs in a transfection assay.

The fate of G.T mismatches and frameshifts, present at the 3'-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G.T mismatches in the middle of the duplex region or at the 3'-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G.T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i-mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3'-terminal G.T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G.T mismatches are degraded by the 3'-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3'-terminal mispairs. Single, double and triple T----C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T----C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3'-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay.

Bacterial Proteins↗

PubMed1991

Genetic assay of misincorporation.

A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3'-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.

Bacteriophages↗

PubMed1991

Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli.

Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G.C base pairs or 5'-pyrimidine, 3'-purine nearest neighbors, although considerable site-to-site variation in the occurrence of base substitutions was seen, even within identical nearest neighbor contexts.

Base Sequence↗

PubMed1988

Gene synthesis technology: recent developments and future prospects.

Gene synthesis is a potentially powerful tool in molecular biology that has not yet reached widespread use because of the relatively high cost and labor-intensive nature of the process. This paper reviews some recent technological developments and current research activities of this laboratory which promise to greatly reduce the cost of gene synthesis and to increase the speed and efficiency of the process. We recently developed an improved device for "segmented" synthesis of oligonucleotides, which utilizes porous Teflon wafers containing derivatized controlled pore glass supports to simultaneously synthesize up to 100 different DNA sequences. The stepwise coupling efficiency with the "wafer synthesis device" is as high as that attained with current automated "gene machines" producing 1-4 oligonucleotides at a time, whereas the reagent usage is only 20-50% that of the current DNA synthesizers. At present, we are optimizing the conditions for rapid, efficient assembly of genes on a solid-phase support, wherein ordered, stepwise annealing/washing is performed to segmentally elongate a "starting" oligonucleotide attached to a solid-phase support. We expect that the wafer synthesis device (operated at reduced scale of synthesis), together with solid-phase gene assembly, will permit the synthesis and assembly of an average size gene (1 kb) in one week at a cost of less than $1000. These developments should make gene synthesis a routine and powerful tool in molecular biology.

Biotechnology↗