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P F Little

Publications and source records attributed to P F Little.

At least 19 recordsLinked to original sources
PubMed1992

High-resolution localization of 69 potential human zinc finger protein genes: a number are clustered.

In this study, we describe the identification and partial characterization of 101 potential human zinc finger protein genes (ZnFPs). These sequences were isolated by hybridization of cosmids, obtained from mouse-human cell lines enriched for chromosome 11p, with an oligonucleotide specific for the "link" sequence between contiguous zinc fingers. Sixty-nine of these cosmids were regionally localized to human prometaphase chromosomes by in situ hybridization. The localization of these cosmids suggests that a number of finger protein genes occur in linked clusters. Their assignment to chromosomes 3p, 11p, 19p, 19qter, 20p, and 21q makes them valuable as markers or "candidate" genes for diseases associated with these chromosome regions.

Amino Acid Sequence↗
PubMed1992

Isolation and expression of linked zinc finger gene clusters on human chromosome 11q.

Proteins that share conserved "zinc finger" motifs represent a class of DNA-binding proteins that have been shown to play a fundamental role in regulating gene expression and to be involved in a number of human hereditary and malignant disease states. We have isolated, characterized, and mapped zinc finger-encoding genes specific to human chromosome 11q to investigate their possible association in the molecular pathogenesis of several disease loci mapped to this chromosome. An arrayed chromosome 11q cosmid library was screened using a degenerate oligonucleotide corresponding to the H/C link consensus sequence of the Drosophila Kruppel zinc finger gene, resulting in the isolation of six putative zinc finger genes. Three of the genes (ZNF123, ZNF125, and ZNF126) were analyzed and shown to contain tandemly repeated zinc finger motifs of the C2-H2 class. All three novel genes were found to be expressed in normal adult human tissues, although the tissue-specific pattern of expression differs markedly. Isolated zinc finger genes were regionally mapped on chromosome 11 using fluorescence in situ suppression hybridization and demonstrated clustering of the genes at 11q13.3-11q13.4 and 11q23.1-11q23.2. Analysis of in situ hybridization to interphase nuclei demonstrated a maximum distance of 1 Mb separating distinct finger genes. This analysis defines two linked multigene families of zinc finger genes to chromosome bands associated with a high frequency of specific translocations associated with malignancies.

Amino Acid Sequence↗
PubMed1991

A cluster of related zinc finger protein genes is deleted in the mouse embryonic lethal mutation tw18.

We report that a number of related zinc finger protein genes are closely linked on mouse chromosome 17. At least four of these genes are transcribed in the 8.5-day postcoitum embryo and are deleted in the t complex early acting embryonic lethal mutation tw18. We have evidence that additional finger protein genes are located in this region. These findings demonstrate that related finger protein genes can be clustered in the murine genome and identify genes that may be considered as candidates for the tw18 mutation.

Base Sequence↗
PubMed1991

The mouse Col2a-1 gene is highly conserved and is linked to Int-1 on chromosome 15.

Type II collagen is the major extracellular matrix component of cartilage and correct expression of the alpha 1(II) collagen gene is important for vertebrate skeletal development. In order to provide the basis for studying the control of type II collagen gene expression in embryogenesis and in mouse models of human connective tissue disease, the complete mouse Col2-a1 gene has been isolated in a single cosmid clone, cosMco1.2, and partially characterized. The gene is approximately 30 kb and is highly conserved in exon/intron structure and nucleotide and amino acid sequence (greater than 80% homology) when compared with the human, rat, bovine and chicken equivalents. A high degree of conservation was also found in the 5' flanking region of the rat, human and mouse alpha 1(II) collagen genes, including the presence of several G + C and C + T rich, direct repeat motifs. The sites of transcription start, termination codon and polyadenylation have also been identified. Unlike chicken, bovine and human, where polyA attachment is at a single site, for the mouse Col2a-1 gene two polyadenylation sites are utilized. Col2a-1 has also been localized by interspecies backcross analysis to the central portion of mouse Chromosome (Chr) 15, approximately 8 centiMorgans (cM) proximal of Int-1 and 18 cM distal of Myc. Col2a-1 is therefore included in a linkage group which is conserved on human Chr 12q.

Amino Acid Sequence↗
PubMed1990

Isolation and regional localisation of DNA sequences from a human chromosome 11-specific cosmid library.

A cosmid library has been prepared in the lorist-B vector from a mouse/human somatic cell hybrid containing region 11q23-11pter as the only human component. This chromosome region is stably maintained in the hybrid as a result of translocation onto one copy of mouse chromosome 13. Individual cosmids containing human DNA were isolated by their ability to hybridise with total human DNA, digested with either HindIII or EcoRI, and 33 individual unique sequences were identified. These fragments were then isolated and subcloned into the bluescribe plasmid vector. Regional localisation of these unique sequences was achieved using a panel of somatic cell hybrids containing different overlapping deletions of chromosome 11. The majority of the 33 mapped sequences derived from the long arm of chromosome 11. Two clones were located within the 11p13-p14 region, which is associated with a predisposition to Wilms' tumour. These probes supplement those already mapped to this chromosome and will assist in the generation of a detailed chromosome 11 linkage map.

Animals↗
PubMed1989

A cosmid clone map derived from a small region of human chromosome 11.

We have used cosmid "fingerprinting" to construct an overlapping DNA clone "map" of the human DNA in a mouse/human hybrid cell line, E65-9, that contains about 4 x 10(6) bp, including the H-Ras gene, as its human component. We have additionally used 32P-labeled RNA probes to establish linkage of particular sets of clones, and the final map comprises about 300,000 bp and is contained in three nonoverlapping segments. The reasons for failure to close the gaps by direct probing are discussed. We have developed techniques to search for rare cutting restriction enzyme cleavage sites in large numbers of cloned DNAs and have positioned sites for EagI and BssHII on our clone map. The methods we used are capable of considerable scale-up and are currently being applied to the short arm of human chromosome 11.

Animals↗
PubMed1988

Two homologous oncogenes, HST1 and INT2, are closely located in human genome.

Pulsed field gel electrophoresis and Southern blot analysis showed that the human oncogenes, HST1 and INT2, which code for proteins homologous to fibroblast growth factors, are less than 45 kb apart on the long arm of chromosome 11. Moreover, analysis of two overlapping cosmid clones, one containing INT2 and the other HST1 sequences, showed that HST1 is located about 35 kb downstream of INT2 in the same transcriptional orientation. The observed close proximity of the INT2 and HST1 genes may provide important insight on the origin and regulation of expression of these related genes.

Blotting, Southern↗
PubMed1988

Smart2, a cosmid vector with a phage lambda origin for both systematic chromosome walking and P-element-mediated gene transfer in Drosophila.

We describe a new phage-lambda-replicon-based cosmid vector suitable for both chromosome walking and P-element-mediated transformation in Drosophila. Its unique BamHI cloning site is flanked by the promoters for the SP6 and T7-encoded RNA polymerases, permitting the synthesis of probes complementary to the ends of the cloned inserts for library screening. The selectable marker is tet for bacterial cell transformation and neo for Drosophila transformation expressed under the control of the Drosophila hsp70 promoter.

Animals↗
PubMed1987

Analysis of thymidylate synthase gene amplification and of mRNA levels in the cell cycle.

We report that the gene for thymidylate synthase (TS) is amplified in the mouse cell line L1210:C15 that was selectively grown in increasing concentrations of the competitive inhibitor of thymidylate synthase, CB3717. The gene is amplified 50-fold compared to the parental cell line. Amplification has not been accompanied by any major rearrangements, and the increase in gene copy number is reflected in elevation of thymidylate synthase mRNA levels. The amplification is relatively stable as there was only a 2- to 3-fold decrease in the number of amplified TS genes when cells were grown in the absence of selection for 375 generations. We also observe a 30- to 40-fold increase in number of copies of the dihydrofolate reductase gene with 7-fold elevation of the RNA product, and we suggest that this may be due to cross-inhibition of dihydrofolate reductase by CB3717. Thymidylate synthase mRNA levels in L1210 and L1210:C15 show no variation within the different phases of the cell cycle but are significantly reduced during quiescence.

Animals↗
PubMed1987

Physical and genetic analysis of cosmids from the vicinity of the cystic fibrosis locus.

Cosmid libraries have been constructed from DNA of somatic cell hybrid cell lines, each containing a fragment of human chromosome seven and including sequences closely linked to cystic fibrosis (CF). Cosmids containing human DNA as insert were isolated from the library. Three cosmids, when used as probes to total genomic DNA, detected polymorphic loci, each of which was shown to be in strong linkage disequilibrium with CF. Restriction endonuclease digestion of cosmid clones and use of a new, rapid method of chromosome walking based on competitive hybridisation of cosmid inserts has allowed identification of several groups of overlapping cosmids ("contigs") from the vicinity of CF.

Animals↗
PubMed1987

Genomic clone of hst with transforming activity from a patient with acute leukemia.

We have previously reported the identification of a novel transforming gene, hst, in DNA samples taken from human stomach cancers and a noncancerous portion of stomach. Five clones, containing the genomic hst gene, were isolated from a human cosmid library constructed from leukocyte DNA from a patient with acute leukemia. All clones possessed transforming activity when transfected to NIH3T3 cells. From one clone, an 8.7 kb BamHI fragment was subcloned into pBR322, and this subclone was active in transforming NIH3T3 cells. This is the first isolation of transformation-competent genomic hst clones directly from a human genomic library, that is, without prior passage through NIH3T3 cells.

Acute Disease↗
PubMed1987

Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: effect on DNA yield and cloned insert frequency.

Transcription terminators have been included in a phage-lambda-replicon-based cosmid vector, Lorist2, to insulate vector genes against transcriptional interference from cloned insert DNA. DNA yields of recombinant clones containing Escherichia coli genomic DNA inserts are more even for Lorist2 than with its progenitor LoristB. However, the terminators provide only a partial reduction in the over-representation of r X DNA-containing clones generally observed in cosmid libraries of Caenorhabditis elegans DNA, suggesting that causes other than transcriptional readthrough into the vector contribute to this problem.

Animals↗
PubMed1987

Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2-encoded protein.

hst is a transforming gene first identified from transformed NIH 3T3 cells that were transfected with DNA of a human stomach cancer. A genomic fragment of hst obtained directly from a human genomic library also has transforming activity. This fragment has a coding sequence identical to that of the hst cDNA prepared from an NIH 3T3 transformant induced by DNA from a stomach cancer. The deduced amino acid sequence of the hst protein is 43%, 38%, and 40% homologous, respectively, to human basic fibroblast growth factor, human acidic fibroblast growth factor, and mouse int-2 protein in selected regions. This suggests that hst encodes a protein related to fibroblast growth factors, which are wide-spectrum mitogens, and to the int-2 protein, a potential oncogene product implicated in murine mammary carcinogenesis.

Amino Acid Sequence↗
PubMed1986

A cosmid vector for systematic chromosome walking.

We describe the construction of a cosmid, LoristB, that contains SP6 and T7 phage-encoded RNA polymerase promoter sequences that are oriented towards and immediately adjacent to HindIII and BamHI cloning sites. We describe techniques for rapidly generating RNA probes from these promoters that must be complementary to the extreme left or right ends of the cloned DNA and can be used for library screening. Probe preparation requires neither prior knowledge of restriction sites nor fragment isolation. We also make extensive use of cos mapping restriction-mapping protocols that we have devised for our cosmid vectors for generation and alignment of steps in a cosmid walk.

Base Sequence↗
PubMed1985

A cosmid vector that facilitates restriction enzyme mapping.

We describe the construction and use of a cosmid vector, loric, which is derived from the phage lambda origin of replication and appears to be more stable than ColE1-derived cosmids. Loric recombinants can be efficiently packaged in vivo to yield 100-300 micrograms of DNA per liter that is linear and has single-stranded cos ends. We call such molecules "phosmids." Phosmid restriction maps can be rapidly generated by labeling either the left or right cos site by annealing on a 32P-labeled oligonucleotide complementary to either cos-L or cos-R. Partial restriction enzyme digestion, agarose gel electrophoresis, and autoradiography are used to size restriction fragments of increasing length, all of which terminate at the labeled cos site. The procedures have been tested by isolating and mapping a region of the H-2 locus of mouse chromosome 17.

Bacteriophage lambda↗