Somatic Cell and Molecular Genetics最新文献

Four-and-a-half LIM domain proteins (FHL) possess four tandem repeats of LIM domain and an extra zinc finger. FHL family LIM proteins are unique when compared with other LIM-only proteins because they possess an odd number of zinc fingers. In this study, the tissue distribution and chromosomal mapping of skeletal muscle LIM protein FHL3 were reported. When the FHL3 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, and virtually no signal could be detected in heart, brain, placenta, lung, liver, kidney and pancreas. Using radiation hybrid technique, FHL3 gene was mapped to the distal end of the short arm of chromosome 1 (123.26 cR from the top of the Chr1 linkage group) and this region (near 1p34) is related to several human malignancies.

The proliferation of suspension cultures of malignant CHO cells was inhibited by 0.5 mM Br-cAMP treatment and restored by its removal. This treatment also inhibited histone H1 phosphorylation completely, reduced histones H2A and H4 phosphorylations, induced DNA degradation, and produced cells containing micronuclei. Agarose gel electrophoresis of the degraded DNA fragments produced a "ladder" pattern confirming these cells were undergoing apoptosis. Cell cycle synchrony experiments demonstrated culture growth inhibition was the result of two different cell cycle-specific processes: [1] arrested cell cycle traverse at a restriction point in mid-G1, and [2] rapid apoptosis following cell division. Br-cAMP did not stop cells in late-G1, S, G2, or M from traversing the cell cycle and dividing, but rather, induced apoptosis following mitosis. The restriction point of Br-cAMP arrest was located in the middle of a wider band of G1 arrest induced by isoleucine deprivation. The cells synchronized in G1 before the restriction point were held in G1-arrest by Br-cAMP and spared apoptotic death. These studies support the further study of cAMP derivatives as agents to induce tumor regression by apoptosis and reverse transformation.

Giemsa-11 or G-11 is a specialized staining technique utilized to (1) differentiate heterochromatic regions of human chromosomes, (2) identify the presence of human chromosomes in human-rodent hybrid cells, and (3) identify human-rodent translocation products in hybrid cells. Earlier procedures, though useful, are problematic and may fail to yield results due to inadequate differentiation between light and dark staining regions. The improved protocol presented here is easy, reliable, and applicable in both clinical and research situations. A discussion of the biology of the staining process is also given.

Testicular germ cell tumors are unusual because they can be cured in over 80% of patients with combination chemotherapy. In order to identify chromosomes carrying genes controlling drug sensitivity, fusions were made between a mouse embryonal carcinoma (EC) cell line, F9, and a human bladder cancer cell line, MGH-UI. In contrast to some previous reports, interspecies hybrids of mouse EC cells with human cells were easy to produce. Six independent hybrids were cloned and grown for 10 further passages and karyotyped. Surprisingly, all the independent hybrids retained approximately 80% of the 40 mouse chromosomes and approximately 80% of the 83 human chromosomes. Despite the positive selection for mouse chromosomes and the absence of selection for human chromosomes, it appears that some of both sets of chromosomes are essential for these hybrids.

The reduced folate carrier (rfc1) gene encodes a protein that is involved in the intracellular accumulation of folates. Point mutations in this gene and alterations resulting in the down regulation of its message are major factors involved in the resistance to antifolate chemotherapeutic compounds. As a framework for understanding the significance of such changes in relation to gene expression and function, in this report we describe the organization of the rfc gene from human lymphoblasts. The gene contains 5 exons (2 to 6) coding for protein. At least four 5' exons, used in a mutually exclusive manner in the production of the rfc message from lymphoblast cells, are spliced to exon 2, which contains the translational start site. "Semi-quantitative" PCR indicates that exon 1 is preferentially used. The major transcriptional start site has been mapped by RACE and RNase protection to a region 109 to 135 base pairs 5' to the start of exon 1. The 5' region of the gene has no TATA box-like sequence but contains several consensus binding sites for transcriptional factors such as SP-1, MZF1, CREB, AP-1, ETS, GATA-1 and GATA-2. The overall organization of the human gene is similar to that of the hamster and mouse genes.

Telomere repeat binding factor 2 (TERF2) is one of two recently cloned mammalian telomere binding protein genes. TERF2 binds as a dimer with high affinity to the double-stranded TTAGGG telomeric repeat through an evolutionarily conserved myb-type DNA binding domain. TERF2 prevents telomere end-to-end fusion and may be important in maintaining genomic stability. We localized the transcribed TERF2 gene to human chromosome 16q22.1, tightly linked to the EST HUM000S343. The mouse Terf2 gene is situated by itself in a newly defined "bin" on chromosome 8 one crossover distal to Psm10 and Sntb2. Human TERF2 and mouse Terf2 are therefore part of a large evolutionarily conserved linkage group comprised of at least 25 known paralogous genes between human chromosome 16q and mouse chromosome 8.

We have isolated and characterized the cDNA for eZNF, a zinc finger gene expressed in human inner ear, from a kinetically enriched human inner ear cDNA library. The sequence of full length cDNA was determined and its expression pattern characterized. A high degree of homology is shared between eZNF and rat transcription factor Kid-1. It belongs to the C2H2 class of zinc finger genes, contains a Kruppel-associated box (KRAB) domain near the N-terminus, and has consensus sites for phosphorylation. The gene is expressed in kidney and inner ear structures of mouse and human as determined by Northern blot analysis. In situ hybridization was used to demonstrate specific expression of the mouse eZNF homologue in epithelial layers of the saccule, semicircular canals, and the cochlea of newborn mice. The genomic clone corresponding to the cDNA was isolated and used for fluorescence in situ hybridization to localize it to human chromosome 5qter. The identification of genes expressed in human inner ear by representational difference analysis, their chromosomal location, and expression pattern of their homologues in developing mouse inner ear comprise a strategy that can potentially identify genes important in hearing and deafness.

The fur gene encodes the endoprotease, furin. We recently demonstrated mutations in both fur alleles in the mutant Chinese hamster ovary (CHO)-K1 strain, RPE.40, and hypothesized that these mutations were responsible for the endoprotease-deficient phenotype of these cells. We now present the structural and functional properties of three protein products derived from the mutant fur alleles. None of these protein products were able to process the precursor to von Willebrand factor, which is processed by wild-type furin. Pro-protein processing activity initially attributed to one of the mutant proteins was due to wild-type furin produced inadvertently from one of the expression constructs used in these experiments. None of the mutant proteins exhibited evidence of autocatalysis, consistent with the lack of activity versus the test substrate, and glycosylation patterns suggested at least two of them remained in the endoplasmic reticulum. These results confirm that RPE.40 cells are furin null mutants, as earlier evidence had suggested.

In this study, we have examined the effects of targeting vector configuration and site of vector linearization on the frequency of targeted recombination at the endogenous CHO APRT locus, and have analyzed the types and class distributions of APRT+ recombinants obtained in APRT targeting experiments employing uncut circular, insertion-type (ends-in), and replacement-type (ends-out) configurations of the same pAG7 targeting vector, including configurations produced by introduction of a double-strand break (DSB) at sites either within, or at the 5' or 3' boundaries of APRT targeting homology. Our results suggest that: 1) plasmid-chromosome targeted recombination in mammalian cells may not be stimulated to the same degree by a DSB in the targeting vector as by a DSB in the chromosomal target; 2) recombinant class distributions are highly dependent upon targeting vector configuration; and 3) one-sided invasion mechanisms may play a significant role in homologous recombination in mammalian cells.

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3' end of the human PRM1-->PRM2-->TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3' of the spermatid-expressed PRM1-->PRM2-->TNP2 domain at position 16p13.13.