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Biomedical subjects

R Kucherlapati

Publications and source records attributed to R Kucherlapati.

At least 109 records · Page 6Linked to original sources
PubMed1993

An integrated approach for identifying and mapping human genes.

We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.

Base Sequence↗
PubMed1993

Characterization of two chromosome 12 cosmid libraries and development of STSs from cosmids mapped by FISH.

We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LL12NC01 and LL12NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.

Base Sequence↗
PubMed1993

Gene targeting in mammalian cells by homologous recombination.

Gene targeting by homologous recombination is a genetic tool that permits modification of cellular genes in a precise and predetermined fashion. The methodologies that are currently available permit gene targeting at high efficiency and fidelity. New developments promise large-scale modification of the mammalian genome using these techniques.

Animals↗
PubMed1992

Organization of the human skeletal myosin heavy chain gene cluster.

Myosin is an important structural and enzymatic component of skeletal muscle. Multiple myosin isoforms are encoded by a multigene family and are expressed in different developmental stages and fiber types. In humans and mice, skeletal myosin heavy chain (MYH) genes are clustered on a single chromosome (17p and 11, respectively). Since the structural organization of the gene cluster may affect its expression as well as shed light on MYH genetic alterations, a physical map of the human MYH gene cluster was constructed. Nine yeast artificial chromosomes containing MYH genes were isolated and used to construct a contiguous set (contig) of overlapping yeast artificial chromosomes. This contig encompasses a genetic marker mapped to 17p13.1. Six MYH genes were located within a 500-kilobase segment of human DNA. The order of the genes within this cluster does not correspond to the developmental pattern of expression of individual members.

Base Sequence↗
PubMed1991

A dominant positive and negative selectable gene for use in mammalian cells.

We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusion protein that has TK and NEO protein functions. This and other fusion genes of a similar nature could serve as dominant positive and negative selectable markers in mammalian cells.

Amino Acid Sequence↗
PubMed1989

Single-stranded DNA gaps, tails and loops are repaired in Escherichia coli.

Uniformly methylated heteroduplex plasmids which contained 6 mismatched regions, including loops of 24, 30, 248 and 283 nucleotides, as well as single-stranded gaps and free ends were introduced into a recombination-deficient strain of bacteria, and the products of repair were analyzed. The results indicate that these cells are capable of repairing all of these structures, although with different efficiencies. Repair of single-stranded gaps and free ends, which occurs most efficiently, is always associated with acquisition of information from the uncut strand (unidirectional repair). Regions containing single loops or twin loops were repaired at similar efficiencies. In these cases each of the two strands was capable of acting as the template for repair (bidirectional repair). At sites containing twin or substitution loops, the larger of the loops was removed twice as efficiently as the smaller loop. DNA sequencing of the repaired regions indicated that the repair is precise. The data also suggest that markers separated by only 58 nucleotides do not always segregate together indicating that repair tracts may be relatively short.

Base Composition↗
PubMed1989

Homologous recombination involving small single-stranded oligonucleotides in human cells.

Gene modification by homologous recombination is one of the techniques that may eventually be used in gene replacement therapy. We tested whether small, synthetic single-stranded oligodeoxynucleotides are capable of participating in homologous recombination in human cells. A plasmid carrying a mutant neomycin phosphotransferase (neo) gene was cotransfected with a 40-nucleotide single-stranded oligomer that contained the wild-type neo gene sequence into human cells. Cells expressing neo were selected in the antibiotic G418. These cells contained wild-type molecules, which resulted from recombination between the two molecules. The results indicate that this approach may be useful in correcting or introducing single point mutations into the genomes of mammalian cells.

Base Sequence↗
PubMed1987

Accurate modification of a chromosomal plasmid by homologous recombination in human cells.

We have examined the consequences of modifying mammalian cellular DNA sequences by homologous recombination. A plasmid carrying a 248-base-pair deletion in the neomycin phosphotransferase (neo) gene was introduced into hamster and human cells. The integrated, defective neo gene was used as a target for modification by a second round of transfection with a plasmid carrying a different (283-base-pair) deletion in the neo gene. Recombinants resulting in an intact neo gene were selected by their G418 resistance phenotype. The best ratio of homologous to nonhomologous recombination events was about 1:80. Analyses of the functional neo genes in various independent cell lines establish that simple crossovers (single and double) generated the wild-type neo genes.

Animals↗
PubMed1987

Repair of single-stranded DNA nicks, gaps, and loops in mammalian cells.

We studied the ability of mammalian cells to repair single-stranded nicks, gaps, and loops in DNA duplexes. Heteroduplexes prepared from derivatives of the shuttle vector pSV2neo were introduced into monkey COS cells. After replication, the plasmids were recovered and used to transform Escherichia coli. Plasmid DNA from the recovered colonies was tested for repair at each of six different sites. We observed that mammalian cells are capable of repairing single-stranded gaps and free single-stranded ends most efficiently. Regions containing twin loops were recognized, and one of the loops was excised. Portions of the molecules containing small single loops were also repaired. Markers which were 58 nucleotides apart were corepaired with nearly 100% efficiency, while markers which were 1,000 nucleotides or more apart were never corepaired. The mechanisms involved in heteroduplex repair in mammalian cells seem to be similar to those involved in repairing DNA lesions caused by physical and chemical agents.

Animals↗
PubMed1987

Characterization of an ATP-dependent DNA strand transferase from human cells.

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.

Adenosine Triphosphatases↗
PubMed1986

Sequence homology requirements for intermolecular recombination in mammalian cells.

We have examined the homology requirements for intermolecular recombination between plasmids introduced into human, monkey, and bacterial cells. Variable-size-deletion derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo were constructed. Each of these plasmids was mixed with another pSV2neo plasmid containing a different, nonoverlapping deletion. Recombination was measured in mammalian cells and bacteria by the frequency of reconstruction of an intact neo gene. We observed that 25 base pairs of homologous sequence is sufficient to yield recombinant products, implying that synapsis and homologous pairing can occur with this level of homology. Examination of the products revealed that nonreciprocal recombination played a role in the generation of normal neo genes. In addition coconversion of linked markers was observed. Exonucleolytic action seems to play a role in gene conversion.

Animals↗
PubMed1986

Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

We have examined the ability of single-stranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mp11. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting single-stranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TK+ colonies. From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.

Animals↗
PubMed1985

Homologous recombination between autonomously replicating plasmids in mammalian cells.

The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neoR colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.

Animals↗
PubMed1985

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

Animals↗