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C J Farr

Publications and source records attributed to C J Farr.

16 recordsLinked to original sources
PubMed1995

Generation of a human X-derived minichromosome using telomere-associated chromosome fragmentation.

A linear mammalian artificial chromosome vector will require at least three functional elements: a centromere, two telomeres and replication origins. One route to generate such a vector is by the fragmentation of an existing chromosome. We have previously described the use of cloned telomeric DNA to generate and stably rescue truncated derivatives of a human X chromosome in a somatic cell hybrid. Further rounds of telomere-associated chromosome fragmentation have now been used to engineer a human X-derived minichromosome. This minichromosome is estimated to be < 10 Mb in size. In situ hybridization and molecular analysis reveal that the minichromosome has a linear structure, with two introduced telomere constructs flanking a 2.5 Mb alpha-satellite array. The highly truncated chromosome also retains some chromosome-specific DNA, originating from Xp11.21. There is no significant change in the mitotic stability of the minichromosome as compared with the X chromosome from which it was derived.

Animals↗
PubMed1995

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

Blotting, Northern↗
PubMed1995

The high mobility group transcription factor, SOX4, transactivates the human CD2 enhancer.

A strong T cell-specific enhancer is located 3' to the human CD2 gene. Six sequences within this enhancer are bound by proteins present in T cell nuclear extracts. These sequences share homology with sequences bound by several transcription factors involved in T cell- and lymphoid-specific transcription. The results presented here demonstrate that the human T cell-specific transcription factor, SOX4, is able to bind to one of these regions; further, SOX4 transactivates transcription of a reporter gene via three tandem copies of this sequence. The binding of SOX4 to this site is not via a canonical HMG protein binding sequence, identifying a novel class of binding site for this protein. A second sequence within the CD2 enhancer closely resembles the IL-2 NF-AT site. We show that it is bound by the ets-related factor, Elf1. However, unlike the IL-2 NF-AT sequence, the CD2 NF-AT-like sequence is unable to confer transcriptional inducibility on a reporter gene. Consistent with this result, we show that the observed increase in expression of CD2 protein on the cell surface following T cell activation is a post-transcriptional event.

Antigens, CD↗
PubMed1994

Sandwiching of a gene within 12 kb of a functional telomere and alpha satellite does not result in silencing.

Pericentric heterochromatin and telomeres have been shown to be capable of repressing the expression of genes located in close proximity. The effect of adjacent structural sequences on gene expression will be important in the design of mammalian artificial chromosomes. In the process of using telomere-containing constructs to generate a deletion panel of the long arm of the human X chromosome, several cell lines were produced which appeared by in situ hybridization to be broken in Xq at or near the centromere. After analysis of end clones rescued from these cell lines, only two produced data consistent with breaks in the alpha satellite array without accompanying rearrangements. The mitotic stability of an X chromosome, with at least 750 kb of the alpha satellite array deleted, was compared to controls where the alpha satellite array remained intact. No significant change in the stability of the chromosome was observed, suggesting that the truncated chromosome has a fully functional mitotic centromere. There was no detectable change in the expression of the hygromycin resistance gene, which is located between a functional centromere and telomere, in this cell line. This study indicates that structural elements flanking a mammalian selectable marker do not result in silencing.

Animals↗
PubMed1993

Genetic maps.

The same genetic principles are common to all animals, plants and microorganisms. One consequence of the generality of genetic principles is that methods developed in one organism can often be used in other species. Recent advances in mammalian molecular genetics, genome analysis, and gene mapping have been found to be directly applicable to other animals and even plants. This is facilitating the rapid construction of detailed genetic maps in a wide variety of species. Similar advances are being made in the development of manipulation techniques, which allow the genomic deletion and addition of specific genes as well as more subtle alterations. Armed with detailed maps and precise manipulation techniques it is possible to associate genes with phenotypes, test the function of genes and to unravel complex traits that depend on the interaction of different genes.

Animals↗
PubMed1993

Characterization and mapping of the human SOX4 gene.

The SOX genes comprise a large family related by homology to the HMG-box region of the testis-determining gene SRY. We have cloned and sequenced the human SOX4 gene. The open reading frame encodes a 474 amino acid protein, which includes an HMG-box. The non-box sequence is particularly rich in serine residues and has several polyglycine and polyalanine stretches. With somatic cell hybrids, human SOX4 has been mapped to Chromosome (Chr) 6p distal to the MHC region. There is no evidence for clustering of other members of the SOX1, -2, and -3 or SOX4 gene families around the SOX4 locus.

Amino Acid Sequence↗
PubMed1993

Novel and known protein tyrosine kinases and their abnormal expression in human melanoma.

We have used the polymerase chain reaction and Northern blotting to identify protein tyrosine kinases that may play an important role in the process of melanoma initiation and progression. Degenerate primers from the conserved catalytic domain of tyrosine kinase genes were used to amplify and clone partial cDNA sequences from a human melanoma cell line (DX3-LT5.1) and normal human melanocytes. When the melanoma reaction products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named MEK (for melanocytic kinase). Of the remaining 12 known kinases, only two, ERB-B2 and IGF1-R, have previously been reported in pigment cells. Reaction products from melanocytes included only eight of these 13 sequences. To test for quantitative differences in tyrosine kinase expression between normal and malignant cells, a panel of eight melanoma lines and normal melanocytes was analyzed by Northern blotting. Two tyrosine kinases (JTK-14/TIE and TYRO-9) were detected in some melanomas but were not found in normal melanocytes, whereas others, including MEK, appeared to be overexpressed in some malignant lines. A minority of kinases showed either no change or a reduction in the level of mRNA. Expression of tyrosine kinases varied independently, and individual lines contained various combinations of these enzymes. Our findings are consistent with an increased overall expression of these putative growth factor receptors during melanoma development.

Amino Acid Sequence↗
PubMed1992

Mutational activation of the N-ras oncogene assessed in primary clonogenic culture of acute myeloid leukemia (AML): implications for the role of N-ras mutation in AML pathogenesis.

The number of steps involved in the pathogenesis of acute myeloid leukemia (AML) is unclear. The initiating event would be expected to exist in all leukemic cells, but subsequent events may be subclonal. If several genetic events occur, they may cooperate within the same cell or be alternatively acquired by different subclones. These possibilities cannot be adequately analyzed in DNA prepared directly from patient specimens. In this study, N-ras mutations demonstrable in DNA prepared from peripheral blood of 10 patients with AML were examined in primary in vitro colonies (AML-colony-forming units [CFU]) grown from these patients. Both colonies containing the mutant gene and colonies containing normal allele only were obtained from each patient. The proportion of colonies containing no mutant allele varied among patients (5% to 57%). A subset of mutation containing colonies appeared to have lost the normal allele in nine of 10 AML cases analyzed. In the four cases with two N-ras mutations, the two mutations were found to exist in different subclones. In these cases, macroscopic colonies (AML-MCFU) were also obtained using an assay system designed to select for earlier clonogenic cells than in the AML-CFU assay. The N12cys mutation in AML10 was found in the CFU, but not in the MCFU, and the N12asp mutation in AML43 was found in the MCFU, but not in the CFU. These results suggest that N-ras mutation is a postinitiation event in AML that contributes to the outgrowth of more malignant subclones. Where two mutations are found in a case of AML, they appear to have been acquired by separate subclones, which may show different degrees of differentiation.

Base Sequence↗
PubMed1992

Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis.

Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.

Animals↗
PubMed1988

Activated ras oncogenes in human thyroid cancers.

Human thyroid epithelial (follicular) cells give rise to two malignant tumors--"follicular" carcinomas, which metastasize almost exclusively via the bloodstream, and "papillary" carcinomas, which metastasize predominantly via lymphatics (Williams, E. D. In: W. Duncan (ed.), Recent Results in Cancer Research: Thyroid Cancer, pp. 47-55. Berlin: Springer-Verlag, 1980). We have investigated whether this contrast in biological behavior might be associated with different patterns of oncogene activation. DNA transfection analysis of five follicular and ten papillary cancers indeed showed a statistically significant difference in the pattern of genes responsible, activated ras oncogenes being found in 80% of follicular tumors but only 20% of papillary tumors. In addition, in follicular cancers we have found activation of all three ras oncogenes (H-ras, K-ras, and N-ras), the first time that this has been demonstrated in a primary human tumor type (as opposed to cell lines). We suggest therefore that ras activation may be an important determinant of metastatic capability in these epithelial cancers.

Animals↗
PubMed1988

Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes.

In vitro DNA amplification followed by oligonucleotide dot blot analysis were used to study RAS gene mutations in acute myeloid leukemia (AML). Fifty-two presentation AML DNAs were screened for mutations in codons 12, 13, and 61 of NRAS and in codons 12 and 61 of KRAS and HRAS. Fourteen (27%) contained mutations--all in NRAS and predominantly in codon 12. The most common amino acid substitution identified was of glycine by aspartic acid at codon 12 (7/18), with a G----A transition being the most common base change (11/18). No particular correlation was observed between disease subtype and the incidence or type of NRAS mutation. In DNA samples from four patients, 2 NRAS mutations were found to coexist. NIH 3T3 focus-formation assays revealed that in each case the mutations were present in different NRAS alleles. We also report the absence of a mutated RAS gene in relapse DNAs of four patients in which a RAS oncogene had been detected at presentation. These observations suggest that RAS mutations arise as part of the evolution of neoplastic transformation.

DNA, Neoplasm↗
PubMed1988

A study of ras gene mutations in colonic adenomas from familial polyposis coli patients.

Using an in vitro amplification step (polymerase chain reaction) followed by oligonucleotide dot blot analysis, DNA samples from 29 familial polyposis coli patients (75 polyp-derived and 26 'normal' colon samples with no epithelial atypia) were screened for the presence of K-, N-, and H-ras mutations. Only 5 polyps contained ras mutations (7%)--all in K-ras codon 12. In each case 'normal' colon DNA was available and found to be negative in this assay. We also report the detection of K-ras codon 12 mutations in a stably non-tumorigenic immortal adenoma-derived cell line, PC/AA, and in a tumorigenic colorectal carcinoma cell line, PC/JW. Both epithelial cell lines were derived from different FPC patients. An activated K-ras gene was also found in cell line S/AN, isolated from a sporadic villous adenoma. These results provide further evidence that there are common molecular events involved in sporadic and hereditary colorectal carcinogenesis and that K-ras mutations can precede the development of malignancy. To our knowledge PC/AA is the first reported example of a human cell line bearing a mutant ras gene that is not tumorigenic and shows that the presence of an activated ras gene even in an immortal human cell line is insufficient for malignancy.

Adenoma↗
PubMed1987

ras gene activation in a minor proportion of the blast population in acute myeloid leukemia.

DNAs from 22 acute myeloid leukaemia (AML) patients were screened for activated transforming genes using NIH3T3 transfection followed by assay for tumor formation in Nude mice. In four samples an activated N-ras oncogene, and in two samples an activated Ha-ras oncogene were detected in transfectants. Synthetic oligonucleotide probes were used to characterise the mutations in the ras genes. Three samples were found to be mutated to N-ras codon 12 ASP, one to N-ras codon 13 ASP and two to Ha-ras codon 12 VAL. When the corresponding AML DNAs were screened using direct gel hybridisation, the mutant ras genes were detectable in only one case. In two AML samples (82 and 84) with very low percentage blasts (3% and 19%), the absence of mutant ras signal from direct gel hybridisation may be due to the lack of sensitivity of this technique in detecting activated ras in such small fractions of the total DNA. These results illustrate the sensitivity of the in vivo tumour assay in detecting activated ras. When DNA from one of the remaining three AML DNAs was amplified using the polymerase chain reaction method, the mutation present in the transfectant was detected. These findings suggest that even in AMLs with high percentage blasts (40%, 70% and 90%), cells containing mutant ras may comprise only a minor proportion of the major leukaemic clone.

Blast Crisis↗