Chromosomal sublocalization of the transcribed human telomere repeat binding factor 2 gene and comparative mapping in the mouse.

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.

Organization and expression of human telomere repeat binding factor genes.

The ends of mammalian chromosomes terminate in structures called telomeres. Recently a human telomere repeat binding factor (TRF1) that binds the vertebrate TTAGGG telomeric repeat in situ was isolated by Chong et al. (1). TRF1 regulates telomere length (2), which is often altered in cancer cells. To understand their genetic organization, TRF1 genes were localized to human chromosomes 13cen, 21cen, and Xq13 by analysis of human monochromosomal hybrids, and by fluorescent in situ hybridization. We also confirmed the recent localization of a human TRF1 gene to chromosome 8, and provide evidence that this locus is alternatively spliced. In contrast to the TRF1 genes on chromosomes 8 and X, the chromosomes 13 and 21 TRF1 genes contained a 60 bp deletion in the coding region. The results suggest that two distinct forms of TRF1 are expressed and that the TRF1 gene family includes at least three pseudogenes whose dispersal in the human genome may have occurred via cDNA intermediates.

Amelioration of ischemia/reperfusion injury in isolated rats hearts by the ATP-sensitive potassium channel opener BMS-180448.

OBJECTIVE: Aims of this study were: (1) Evaluate by morphology and specific physiological and biochemical parameters, the protective effects of the cardioselective ATP-sensitive potassium channel opener BMS-180448 on ischemic/reperfused isolated rat heart, and (2) Determine the earliest time point ischemia-induced myocardial injury is observed by light microscopy. METHODS: Hearts from Sprague-Dawley rats were perfused on a Langendorff apparatus. After equilibration, hearts were treated with BMS-180448 (10 micro M) or vehicle (0.04% DMSO) for 10 min before the onset of ischemia. Four hearts/group were collected following 10, 18, or 25 min of ischemia. A nonischemic control group was also evaluated. Following 25 min of ischemia, another set of hearts was reperfused with oxygenated Krebs-Hensleit solution and allowed to recover for 30 min. Light and electron microscopic changes of the myocardium were semi-quantitatively evaluated together with physiological (i.e., heart rate, left ventricular diastolic pressure, time to contracture formation) and biochemical (i.e., lactate dehydrogenase, LDH, release) endpoints. RESULTS: Cardioprotective effects of BMS-180448 following ischemia/reperfusion consisted of a reduced rate of contracture formation, reduced LDH release, and enhanced recovery of contractile function during reperfusion (P < 0.05). Light microscopic evidence of myocardial damage was detected following 18 min of ischemia. Morphological changes in ischemic/reperfused hearts included interstitial edema, myofiber degeneration, and hypercontraction band formation. Ultrastructurally, swollen myofibrils, swollen mitochondria with disrupted cristae and electron-dense deposits, myofibrillar lysis, and contraction bands, were observed. Light and electron microscopic severity scores were significantly less (P < 0.05) in BMS-180448-treated hearts at the 25 min ischemic time point and in reperfused hearts, as compared to similarly-treated vehicle hearts. CONCLUSIONS: BMS-180448 ameliorates morphological evidence of ischemia/reperfusion myocardial damage in the isolated rat heart model, in agreement with physiological and biochemical parameters.

Chromosome microdissection identifies cryptic sites of DNA sequence amplification in human ovarian carcinoma.

DNA sequence amplification contributes to the multistep process of carcinogenesis, and overexpression of amplified genes has been shown to contribute to the malignant phenotype. Cytogenetic analyses of human tumor cells, including ovarian malignancies, frequently show cytological evidence of DNA amplification in the form of double minutes and homogeneously staining regions. In this report, we have combined the techniques of chromosome microdissection and fluorescence in situ hybridization (P. S. Meltzer et al., Nat. Genet., 1: 24-28, 1992) to identify the composition and chromosomal origin of seven homogeneously staining regions from seven cases of ovarian cancer. Twelve specific chromosome band regions were identified as amplified including 11q, 12p, 16p, 19p, and 19q. These results provide important insights into the organization of amplified sequences within ovarian malignancies and add further to our recognition of regions likely to harbor genes important to the development or progression of ovarian cancer.

Pharmacokinetics of hydroxyurea in nude mice.

Extrachromosomal DNA is the predominant form of gene amplification in human tumors. Hydroxyurea (HU) concentrations of 100-150 microM have been promising in vitro for extrachromosomal DNA elimination. The study objective was to determine the HU dose-concentration relationship in nude mice with HU doses from 0 to 200 mg/kg. For HU t1/2 determination, mice were injected with HU 100 mg/kg. A plasma concentration of 159 microM was achieved and a t1/2 of 11.3 min determined. Based on these findings, In vivo elimination studies will require frequent administration of HU to maintain plasma concentrations from 100 to 150 microM.

Double minutes are frequently found in ovarian carcinomas.

Double minutes (dmins) are acentric chromosomal-like entities that are important in the etiology of cancer because they are known to harbor amplified oncogenes and drug resistance genes. Because dmins can be unequally partitioned at mitosis they have the ability to confer genetic diversification rapidly. Selective pressures operative in vitro may be quite different than those in vivo; therefore, tumor cells which harbor dmins could be selected against during short-term in vitro propagation. We wanted to determine the incidence of dmins in human ovarian cancer cells obtained from fresh ovarian specimens with an absolute minimum of culture time (6-24 hours). In "direct" chromosomal preparations obtained from these clinical specimens we found dmins present in 88% of these samples. This remarkable finding that dmins are found so frequently in ovarian cancers underscores the importance of gene amplification in human tumor biology. Therefore, the presence of dmins in patient specimens indicates that these unstable genetic elements may play a significant role in the maintenance or progression of malignancy.

Evidence of gene amplification in the form of double minute chromosomes is frequently observed in lung cancer.

Amplification of cellular proto-oncogenes, important in tumor progression, has been correlated with a poor clinical outcome in a variety of human tumor types. Amplified genes are observed in two cytogenetically distinct entities, double minutes (DMs) and homogeneously staining regions (HSR). We examined 54 fresh lung tumor specimens obtained from patients with non-small cell lung cancer for cytogenetic evidence of gene amplification in the form of DMs. The majority of these patients had received no prior treatment. The cells were harvested within 24 hours after receiving the specimens, and the slides were stained with Giemsa to specifically look for DMs. We found DMs in 24 of 31 (77%) specimens that exhibited metaphase spreads. Similar incidences of DMs were found when histologic cell types, primary vs. non-primary tumors, and specimens from patients with prior treatment vs. no prior treatment were compared. Therefore, DMs occur frequently in non cultured lung tumor cells, providing evidence that gene amplification may be an important aspect of tumor behavior in patients with non-small cell lung carcinoma. Further investigation is warranted to identify the specific tumor-related genes located on these abnormal chromosomes. This also suggests that ongoing efforts to eliminate amplified drug-resistant genes or oncogenes contained on DMs in tumor cells may be relevant in patients with non-small cell lung cancer.

Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity.

Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malignancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as double-minute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms.

Chromosomal influence on hybrid cell proliferation.

When normal cells and cancer cells (usually from the same species) are experimentally fused the resultant hybrid cells show loss of the tumorigenic phenotype. To examine the phenotypic phenomenon of growth suppression in hybrid cells in vitro, we examined the doubling times of somatic cell hybrids which contained single or multiple chromosomes derived from another species (inter-species hybrids). In all of the hybrid lines examined, the presence of transferred chromosomes prolonged the cell population doubling times in proportion to the number of such chromosomes found in the hybrid lines. These findings provide additional evidence to support the hypothesis that increasing the genetic burden of cells may reduce the division potential of cells cultured in vitro.

Expression of the human inhibin alpha-subunit gene in preovulatory granulosa-theca cells.

Inhibin, a gonadal peptide that suppresses pituitary follicle-stimulating hormone, with lesser or no effect on luteinizing hormone, has recently been purified and the complementary deoxyribonucleic acid sequences cloned. Inhibin contains two subunits, labeled alpha-subunit and beta-subunit. Here we report for the first time the detection of human inhibin alpha-subunit gene expression in preovulatory granulosa-theca cells by Northern analysis. The transcript is the same size as previously reported for human placenta and corpus luteum, suggesting that the same gene is being expressed in all three tissues. These findings are consistent with previously reported Southern analysis of deoxyribonucleic acid, which showed only one copy of the alpha-inhibin gene in the human genome. Thus current data strongly suggest that there is only one copy of the inhibin alpha-subunit gene in the human genome, and this same gene is expressed in granulosa-theca cells, corpus luteum, and placenta.

Male infant with ichthyosis, Kallmann syndrome, chondrodysplasia punctata, and an Xp chromosome deletion.

We report on a male infant with X-linked ichthyosis, X-linked Kallmann syndrome, and X-linked recessive chondrodysplasia punctata (CPXR). Chromosome analysis showed a terminal deletion with a breakpoint at Xp22.31, inherited maternally. This patient confirms the localization of XLI, XLK, and CPXR to this region of the X chromosome and represents an example of a "contiguous gene syndrome." A comparison of the manifestations of patients with CPXR, warfarin embryopathy, and vitamin K epoxide reductase deficiency shows a remarkable similarity. However, vitamin K epoxide reductase deficiency does not appear to be the cause of CPXR. We propose that CPXR may be due to a defect in a vitamin K-dependent bone protein such as vitamin K-dependent bone carboxylase, osteocalcin, or matrix Gla protein.

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase.

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.

Chromosomal localization of human lactotransferrin gene (LTF) by in situ hybridization.

Lactotransferrin (LTF) is an important member of the transferrin family of proteins. These proteins play an essential role in the transport of iron in extracellular fluid (Aisen and Listowsky, 1980). Southern blot analysis of mouse-human somatic cell hybrids have localized the LTF gene to region q21----qter of human chromosome 3 (Teng et al., unpublished data). Using the same full-length mouse cDNA probe (2.2 kb), the LTF gene was mapped to human chromosomal bands 3q21----q23 by in situ hybridization. The sublocalization of the LTF gene to 3q21----q23 is in the region of human chromosome 3 where the gene loci of transferrin and transferrin receptor have been localized (Yang et al., 1984; van de Rijn et al., 1983).

Human alpha 2-HS-glycoprotein localized to 3q27----q29 by in situ hybridization.

Human plasma protein alpha 2-HS-glycoprotein (AHSG) is composed of two polypeptide chains, A and B, encoded by a single mRNA. Southern blot analysis of mouse x human somatic cell hybrids has mapped the AHSG gene to human chromosome 3 in the region 3q21----qter (Lee et al., 1987). Using a recombinant plasmid containing a 1,538 bp insert spanning the entire AHSG coding region, AHSG was localized to chromosomal bands 3q27----q29 by in situ hybridization.

Distally based vastus lateralis muscle flap for coverage of wounds about the knee.

The blood supply to the vastus lateralis muscle has been evaluated by dye injection techniques in fresh cadaver dissections. The main dominant blood supply is the descending branch of the lateral femoral circumflex artery. Vascular contributions from distal perforators of the superficial femoral artery, the superior geniculate artery, fill the main vascular pedicle in a retrograde fashion. Latex staining is observed consistently in the proximal third of the muscle. Five patients are presented in whom the distally based vastus lateralis muscle flap was successfully used to cover defects above the knee. Superficial muscle necrosis is a complication of this operation but has not precluded its usefulness. It is anticipated that this flap will be useful in the armamentarium of reconstructive surgeons treating such problematic patients.

Human ferritin H and L sequences lie on ten different chromosomes.

In humans, the H (heavy) and L (light) chains of the iron-storage protein ferritin, are derived from multigene families. We have examined the chromosomal distribution of these H and L sequences by Southern analysis of hybrid cell DNA and by chromosomal in situ hybridization. Our results show that human ferritin H genes and related sequences are found on at least seven different chromosomes while L genes and related sequences are on at least three different chromosomes. Further, we have mapped the chromosomal location of expressed genes for human H and L ferritin chains and have found an H sequence which may be a useful marker for idiopathic hemochromatosis.

Chromosome localization of the human renin gene (REN) by in situ hybridization.

Previous studies by Southern blot analysis of human X mouse somatic cell hybrids localized the renin gene to region p21----qter of human chromosome 1. Using a DNA insert encoding exons 2-5, the renin gene was mapped to human chromosome bands 1q25----q32 by in situ hybridization. The sublocalization of the renin gene will facilitate subsequent detailed linkage analysis of human chromosome 1.

Characterization, mapping, and expression of the human ceruloplasmin gene.

Ceruloplasmin (CP) is a copper-binding protein in vertebrate plasma. It is the product of an intragenic triplication and is composed of three homologous domains. Oligonucleotide probes constructed according to published amino acid sequences were used to identify cDNA clones encoding human CP. Two clones, CP-1 and CP-2, differed from each other by the presence or absence, respectively, of a deduced sequence of four amino acids. The two clones provided 81% of the sequence encoding CP. Comparison of the nucleotides of the three domains of the CP coding sequence revealed internal domain homology with identity of sequences ranging from 50.1% to 56%. The nucleotide sequence of CP-2 cDNa was compared to that of a homologous human protein, clotting factor VIII, and was found to be 48% identical overall. The CP gene was mapped to human chromosome 3 by somatic-cell-hybrid analysis and to 3q25 by in situ hybridization; however, sites of hybridization to DNA on other chromosomal sites suggested additional CP-like DNA sequences in the human genome. A DNA polymorphism was detected with CP cDNA after endonuclease digestion of human DNA by Pst I. CP mRNA was detected in human liver, macrophages, and lymphocytes by in situ histohybridization.

Chromosomal localization of group-specific component by in situ hybridization.

Group-specific component (GC), an alpha 2-globulin plasma protein synthesized primarily in the liver, is the major vitamin D-binding protein in plasma. It has two common phenotypes, GC1 and GC2, which appear in all human populations. Using the cDNA insert containing the entire coding sequence of GC2, the GC gene was mapped to human chromosomal bands 4q13----q21.1 by in situ hybridization.