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Biomedical subjects

R Espinosa

Publications and source records attributed to R Espinosa.

At least 109 records · Page 6Linked to original sources

HEN1 and HEN2: a subgroup of basic helix-loop-helix genes that are coexpressed in a human neuroblastoma.

An important family of regulatory molecules is made up of proteins that possess the DNA-binding and dimerization motif known as the basic helix-loop-helix (bHLH) domain. The bHLH family includes subgroups of closely related proteins that share common functional properties and overlapping patterns of expression (e.g., the MyoD1 and achaete-scute subgroups). In this report we describe HEN1 and HEN2, mammalian genes that encode a distinct subgroup of bHLH proteins. The HEN1 gene was identified on the basis of cross-hybridization with TAL1, a known bHLH gene implicated in T-cell acute lymphoblastic leukemia. In situ fluorescence hybridization was used to localize the human HEN1 gene to chromosome band 1q22. HEN1 and HEN2 are coexpressed in the IMR-32 human neuroblastoma cell line, and they encode highly related proteins of 133 and 135 residues, respectively, that share 98% amino acid identity in their hHLH domains. These data imply that the bHLH protein subgroup encoded by HEN1 and HEN2 may serve important regulatory functions in the developing nervous system.

Amino Acid Sequence↗

Fluorescence in situ hybridization: a sensitive method for trisomy 8 detection in bone marrow specimens.

Trisomy 8 is a common anomaly in bone marrow (BM) cells of patients with myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), or acute nonlymphocytic leukemia (ANLL). We studied the efficacy of fluorescence in situ hybridization (FISH) detection of trisomy 8 in patients with MPD, MDS, or ANLL using directly labeled fluorescent alpha-satellite and whole chromosome paint (WCP) DNA probes specific for chromosome 8. Using FISH, we analyzed interphase nuclei and metaphase spreads from randomized series of BM specimens from normal individuals and patients with varying proportions of trisomy 8 as determined by conventional cytogenetic analysis. The BM of all normal donors contained less than or equal to 2.0% nuclei with 3 interphase FISH signals and less than or equal to 1 metaphase with 3 WCP FISH signals. Ninety-five percent and 98% of BM specimens with at least two metaphase cells with trisomy 8 by cytogenetic analysis contained greater than 2.0% nuclei with 3 interphase FISH and greater than 2 metaphases with 3 WCP FISH signals, respectively. Thirteen patients had 1 in 20 or 1 in 30 metaphase cells with trisomy 8 by conventional cytogenetic studies. Of these patients, four had greater than 2.0% nuclei with 3 interphase FISH signals. The BM of all four patients contained positive metaphase FISH results. We then studied the usefulness of FISH analysis to detect occult trisomy 8 by analyzing BM nuclei from 144 patients who had MPD, MDS, or ANLL and either 20 normal metaphase cells or an abnormal karyotype without trisomy 8. Seven patients had greater than 2.0% nuclei with 3 interphase FISH signals (range, 2.10% to 3.40%) and six patients had 2 or more cells with trisomy 8 upon metaphase FISH or extensive conventional cytogenetic analysis. Our results show that interphase and metaphase FISH analyses are useful methods to detect trisomy 8 cells in BM specimens, especially for specimens with normal or uncertain conventional cytogenetic results.

Bone Marrow↗

Molecular cloning of the breakpoints of a complex Philadelphia chromosome translocation: identification of a repeated region on chromosome 17.

Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells.

Animals↗

Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.

In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.

Amino Acid Sequence↗

Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity.

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3) are the three members of the neurotrophin family known to exist in mammals. Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4), which shares all of the features found in the mammalian neurotrophins, has been identified in Xenopus and viper. We used sequences specific to the Xenopus/viper NT-4 to isolate a neurotrophin from both human and rat genomic DNA that appears to represent the mammalian counterpart of Xenopus/viper NT-4. Human NT-4 as well as a human NT-4 pseudogene colocalize to chromosome 19 band q13.3. Mammalian NT-4 has many unusual features compared to the previously identified neurotrophins and is less conserved evolutionarily than the other neurotrophins. However, mammalian NT-4 displays bioactivity and trk receptor specificity similar to that of Xenopus NT-4.

3T3 Cells↗

A method for the rapid sequence-independent amplification of microdissected chromosomal material.

We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3' end and a defined sequence at its 5' end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.

Base Sequence↗

Chromosomal localization of the genes encoding the p50/p105 subunits of NF-kappa B (NFKB2) and the I kappa B/MAD-3 (NFKBI) inhibitor of NF-kappa B to 4q24 and 14q13, respectively.

The regulation of expression of a variety of genes involved in immune function, inflammation, and cellular growth control, as well as control of expression of certain viruses such as the human immunodeficiency virus (HIV), is dependent on the transcription factor NF-kappa B. In many cells, NF-kappa B is found in the cytoplasm where it is associated with an inhibitor protein known as I kappa B. Recently the genes encoding the p50 and p65 subunits of NF-kappa B, as well as one form of I kappa B/MAD-3 (NFKBI), have been cloned. As part of our goal to determine the chromosomal organization of members of the REL/NFKB family, as well as their inhibitors, we localized the NFKBp50/p105 (NFKB2) and I kappa B/MAD-3 (NFKBI) genes to human chromosome bands 4q24 and 14q13, respectively.

Chromosome Mapping↗

A physical and genetic linkage map of the distal long arm of human chromosome 5.

By in situ hybridization of probes for three cloned genes and eight genetically-linked polymorphic DNA markers, we have prepared a physical map of the distal long arm of chromosome 5. These results, together with the localizations of 11 genes and the genetic linkage map reported previously by us and by other investigators, represent a map that spans 55 cM.

Chromosome Mapping↗

Increased genetic instability of the common fragile site at 3p14 after integration of exogenous DNA.

We determined previously that the selectable marker pSV2neo is preferentially inserted into chromosomal fragile sites in human x hamster hybrid cells in the presence of an agent (aphidicolin) that induces fragile-site expression. In contrast, cells transfected without fragile-site induction showed an essentially random integration pattern. To determine whether the integration of marker DNA at fragile sites affects the frequency of fragile-site expression, the parental hybrid and three transfectants (two with pSV2neo integrated at the fragile site at 3p14.2 [FRA3B] and specific hamster fragile sites [chromosome 1, bands q26-31, or mar2, bands q11-13] and one with pSV2neo integrated at sites that are not fragile sites) were treated with aphidicolin. After 24 h the two cell lines with plasmid integration at FRA3B showed structural rearrangements at that site; these rearrangements accounted for 43%-67% of the total deletions and translocations observed. Structural rearrangements were not observed in the parental cell line. After 5 d aphidicolin treatment, the observed excess in frequency of structural rearrangements at FRA3B in the cell lines with pSV2neo integration at 3p14 over that in the two lines without FRA3B integration was less dramatic, but nonetheless significant. Fluorescent in situ hybridization (FISH) analysis of these cells, using a biotin-labeled pSV2neo probe, showed results consistent with the gross rearrangements detected cytogenetically in the lines with FRA3B integration; however, the pSV2neo sequences were frequently deleted concomitantly with translocations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

TAL2, a helix-loop-helix gene activated by the (7;9)(q34;q32) translocation in human T-cell leukemia.

Tumor-specific alteration of the TAL1 gene occurs in almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL). We now report the identification of TAL2, a distinct gene that was isolated on the basis of its sequence homology with TAL1. The TAL2 gene is located 33 kilobase pairs from the chromosome 9 breakpoint of t(7;9)(q34;q32), a recurring translocation specifically associated with T-ALL. As a consequence of t(7;9)(q34;q32), TAL2 is juxtaposed with sequences from the T-cell receptor beta-chain gene on chromosome 7. TAL2 sequences are actively transcribed in SUP-T3, a T-ALL cell line that harbors the t(7;9)(q34;q32). The TAL2 gene product includes a helix-loop-helix protein dimerization and DNA binding domain that is especially homologous to those encoded by the TAL1 and LYL1 protooncogenes. Hence, TAL2, TAL1, and LYL1 constitute a discrete subgroup of helix-loop-helix proteins, each of which can potentially contribute to the development of T-ALL.

Amino Acid Sequence↗

Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias.

Recurring chromosomal translocations involving chromosome 11, band q23, have been observed in acute lymphoid leukemias and especially in acute myeloid leukemias. We recently showed that breakpoints in four 11q23 translocations, t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13.3), were contained within a yeast artificial chromosome clone bearing the CD3D and CD3G gene loci. We have identified within the CD3 yeast artificial chromosome a transcription unit that spans the breakpoint junctions of the 4;11, 9;11, and 11;19 translocations, and we describe two other, related transcripts that are upregulated in the RS4;11 cell line. We have named this gene MLL (myeloid/lymphoid, or mixed-lineage, leukemia.

Adult↗

Preferential integration of marker DNA into the chromosomal fragile site at 3p14: an approach to cloning fragile sites.

Fragile sites are specific regions of chromosomes that are prone to breakage. In cells cultured under conditions that induce fragile site expression, high levels of inter- and intrachromosomal recombination have been observed involving chromosomal bands containing fragile sites. To determine whether expression of specific fragile sites would facilitate preferential integration of exogenous DNA at these recombination hot spots, the vector pSV2Neo was transfected into a Chinese hamster-human somatic cell hybrid containing a derivative chromosome 3 as its only human component. Chromosome 3 contains a common fragile site at band 3p14.2 (FRA3B) that is induced by aphidicolin. Both cells induced to express FRA3B and the uninduced control cells were transfected with the pSV2Neo selectable plasmid. In situ hybridization of a biotin-labeled pSV2Neo probe to metaphase chromosomes revealed one to three integration sites in each stably transfected clone. Four of 13 clones transfected under conditions of FRA3B induction showed integration of pSV2Neo at 3p14; these clones also showed specific integration into hamster chromosome 1 and a rearranged chromosome characteristic of CHO cells (mar2). The 7 control clones, however, showed an apparently random pattern of pSV2Neo integration. Significant hybridization of pSV2Neo to both FRA3B and Chinese hamster chromosomes 1 and mar2 was seen in 100 cells from pooled colonies transfected after treatment with aphidicolin. These results suggest that preferential integration of marker DNA into human and Chinese hamster fragile sites occurs with exposure to aphidicolin. The nature of the DNA sequences at fragile sites is unknown and, despite a number of approaches, these sequences have not yet been isolated; our procedure may represent an approach to the cloning of fragile sites.

Animals↗

Human and rat brain-derived neurotrophic factor and neurotrophin-3: gene structures, distributions, and chromosomal localizations.

The development and maintenance of the vertebrate nervous system depends upon neuronal survival proteins known as neurotrophic factors. Nerve growth factor (NGF) remains the best characterized neurotrophic molecule. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are two recently cloned neurotrophic factors that are homologous to NGF. Here we describe the molecular cloning of the human and rat genes encoding BDNF, as well as the isolation of the human NT-3 gene. On the basis of comparison of our genomic and cDNA clones with those of previously isolated BDNF and NT-3 genes and cDNAs, we make inferences about the structures of processed transcripts derived from the neurotrophin genes and the protein precursors they encode. We demonstrate that the mature form of BDNF is identical in all mammals examined, and that the same is true of the mature form of NT-3. Furthermore, the respective tissue-distributions and neuronal specificities of NT-3 and BDNF are also conserved among mammals. Finally, we localize the gene encoding human BDNF (gene symbol designated BDNF) to chromosome 11, band p13, and the gene encoding human NT-3 (gene symbol designated NTF3) to chromosome 12, band p13.

Amino Acid Sequence↗

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.

Amino Acid Sequence↗

Localization of the gene encoding insulin-degrading enzyme to human chromosome 10, bands q23----q25.

Insulin-degrading enzyme (IDE) is a cytosolic proteinase involved in the cellular processing of insulin. Using somatic cell hybrid analysis and in situ chromosomal hybridization, we have localized the gene encoding IDE to human chromosome 10, bands q23----q25. The murine Ide gene was previously mapped to Chromosome 19; together, these results suggest that the IDE gene is a member of a conserved syntenic group on human chromosome 10, bands q23----q25 and mouse Chromosome 19.

Animals↗

Mapping chromosome band 11q23 in human acute leukemia with biotinylated probes: identification of 11q23 translocation breakpoints with a yeast artificial chromosome.

Translocations involving chromosome 11, band q23, are frequent recurring abnormalities in human acute lymphoblastic and acute myeloid leukemia. We used 19 biotin-labeled probes derived from genes and anonymous cosmids for hybridization to metaphase chromosomes from leukemia cells that contained four translocations involving band 11q23: t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13). The location of the cosmid probes relative to the breakpoint in 11q23 was the same in all translocations. Of the cosmid clones containing known genes, CD3D was proximal and PBGD, THY1, SRPR, and ETS1 were distal to the breakpoint on 11q23. Hybridization of genomic DNA from a yeast clone containing yeast artificial chromosomes (YACs), that carry 320 kilobases (kb) of human DNA including CD3D and CD3G genes, showed that the YACs were split in all four translocations. These results indicate that the breakpoint at 11q23 in each of these translocations occurs within the 320 kb encompassed by these YACs; whether the breakpoint within the YACs is precisely the same in the different translocations is presently unknown.

Adult↗