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Biomedical subjects

R Espinosa

Publications and source records attributed to R Espinosa.

At least 55 records · Page 3Linked to original sources
PubMed1996

Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases.

Loss of a whole chromosome 7 or a deletion of the long arm, del(7q), are recurring abnormalities in malignant myeloid diseases. To determine the location of genes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia. Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q. The majority of patients (65 of 81 [80%]) had proximal breakpoints in bands q11-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22. The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33. To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22. We determined that the smallest overlapping deleted segment is contained in a well-defined YAC contig that spans 2 to 3 Mb. These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation.

Acute Disease↗
PubMed1996

Direct cloning of DNA sequences from the common fragile site region at chromosome band 3p14.2.

Despite several lines of evidence suggesting that common chromosomal fragile sites are biologically important as hot spots for recombination, their structure remains unknown. We showed previously that the plasmid pSV2neo preferentially integrates into bands containing fragile sites in cells transfected under conditions of fragile site induction. Here we report the isolation and characterization of the DNA sequences from two such independent integrations into 3p14.2, a common fragile site (FRA3B). These FRA3B region sequences were shown to lie within a 1330-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement. The two integration sites are 10 kb apart, but each integration is associated with a deletion. We have constructed a partial genomic contig of the integration sites and deleted regions spanning approximately 85 kb. Analysis of the DNA sequences immediately surrounding the plasmid integrations revealed no known coding sequences or repeat structures resembling the (CGG)n motif characteristic of the rare fragile sites. In addition, by Southern blotting analysis, none of the phage clones isolated from the FRA3B region were found to contain CGG repeats. Fluorescence in situ hybridization analysis of genomic clones from this contig to metaphase cells induced to express breaks demonstrated hybridization adjoining the chromosome breaks, and occasionally the hybridization signal spanned the break. The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb). Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites.

Aphidicolin↗
PubMed1996

A yeast artificial chromosome-based map of the region of chromosome 20 containing the diabetes-susceptibility gene, MODY1, and a myeloid leukemia related gene.

We have generated a physical map of human chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The yeast artificial chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene.

Base Sequence↗
PubMed1996

Analysis of the t(6;11)(q27;q23) in leukemia shows a consistent breakpoint in AF6 in three patients and in the ML-2 cell line.

The t(6;11)(q27;23) is one of the most common translocations observed in patients with acute myeloid leukemia (AML). The translocation breakpoint involves the MLL gene, which is the human homolog of the Drosophila trithorax gene, at 11q23 and the AF6 gene at 6q27. Reverse transcriptase-polymerase chain reaction (RT-PCR) using an MLL sense primer and an AF6 antisense primer detected the MLL/AF6 fusion cDNA from three leukemia patients with the t(6;11) [two AML and one T-acute lymphoblastic leukemia (ALL)] and one cell line. The fusion point in the AF6 cDNA from these cases is identical, regardless of the leukemia phenotype. The ML-2 cell line, which was established from a patient with AML that developed after complete remission of T-cell lymphoma, has retained an 11q23-24 deletion from the lymphoma stage and has acquired the t(6;11) with development of AML. The ML-2 cells have no normal MLL gene on Southern blot analysis, which indicates that an intact MLL gene is not necessary for survival of leukemic cells.

Adolescent↗
PubMed1996

Scaffold attachment regions in centromere-associated DNA.

Due to indications that kinetochore proteins are an integral part of the protein scaffold component of the chromosome (Earnshaw et al. 1984), we chose to map the distribution of scaffold attachment regions (SARs) at centromeres. Using the SAR mapping assay of Mirkovitch et al., Southern blots were prepared and probed with 32P-labeled fragments from the human 1.9 kb centromeric alpha-satellite repeat unit of chromosome 1 or the 1.7 kb centromeric alpha-satellite repeat unit of chromosome 16. Our results demonstrated the presence of one SAR site per 1.9 kb repeat unit in chromosome 1, and every 1.7 kb repeat unit in chromosome 16, separated by regions of small DNA loops over the length of the alpha-satellite regions. We also identified several in vitro vertebrate topoisomerase II and cenP-B consensus sequences throughout the chromosome 1 alpha-satellite region using computer and base ratio analysis, to address the question as to why some alpha-satellite regions are SAR related and others are not. To provide in situ indications of SAR localization in the human genome, SAR DNA and non-SAR DNA were prepared following lithium 3,5-di-iodosalicylate extraction. Sequences protected from DNAse I digestion by SAR proteins, as compared with unprotected DNA that was digested by the enzyme, was labeled with biotin-UTP, hybridized to chromosomal DNA in situ, and then detected with fluorescein-avidin-DCS. Both SAR and non-SAR DNA selectively labeled virtually all centromeric regions of the human metaphase karyotype. Chromosomal arms were less strongly bound by SAR DNA, with a pattern that followed the chromosomal axis. In the more condensed chromosomes an R-banding pattern was evident. In general, labeling patterns produced by both SAR and non-SAR fractions were similar, as expected from the indications that SAR DNAs are heterogenous in sequence and do not form a specific class of sequences. We conclude that centromeric regions of several, possibly all, human metaphase chromosomes are also regions where the chromosomal axis contains loops, smaller in size than in the arms and where attachment sites are concentrated. This clustering of SARs may be responsible in part for the tight chromatin packing associated with the primary constriction of the centromeric region.

Blotting, Southern↗
PubMed1996

The human mitochondrial citrate transporter gene (SLC20A3) maps to chromosome band 22q11 within a region implicated in DiGeorge syndrome, velo-cardio-facial syndrome and schizophrenia.

The gene encoding the human mitochondrial citrate transporter designated SLC20A3 was mapped to chromosome 22 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This assignment was confirmed by fluorescence in situ hybridization to metaphase chromosomes, and the gene was further localized to band 22q11.21. The gene is located in a critical region associated with allelic losses in a variety of clinical syndromes, including DiGeorge syndrome, velo-cardio-facial syndrome and a subtype of schizophrenia.

Abnormalities, Multiple↗
PubMed1996

Paroxysmal atrial tachycardia recorded by Holter monitoring during an episode of fear. A case report.

A twenty-four-year-old, white, athletic woman, free of heart disease, experienced an episode of fear when she was assaulted in the street without physical injury while under-going twenty-four-hour Holter monitoring. She developed an important sympathetic response in which, besides the symptoms characterized by palpitations, chest pain, dyspnea, asthenia, dizziness, nausea, and profuse cold sweating, she had an episode of paroxysmal atrial tachycardia. The causes and mechanism of this not well-documented event in humans are discussed.

Adult↗
PubMed1996

Advances in high resolution SIMS studies of BrdU-labelled human metaphase chromosomes.

The detection of bromine in human metaphase chromosomes labelled with the thymidine-analog BrdU, by imaging Secondary Ion Mass Spectrometry (SIMS) with a high resolution scanning ion microprobe, provides detailed maps of the AT distribution within the chromosomes. Similarly, maps of the emitted CN-molecular ions describe the overall DNA, RNA and protein distribution, details of which are also revealed by maps of the divalent cations Ca+ and Mg+. Base-specific banding patterns (SIMS bands), mimicking the well known G-or Q-bands resulting from conventional staining methods for optical microscopy, are observed in several preparations, more noticeably in mitotic cells at the first cell division, after in situ DNA denaturation, or Giemsa staining. A structured distribution, seemingly related to G/Q-banding patterns, is also observed in the Mg+ and Ca+ maps. The differential label signal intensities between sister chromatids, at the second cell division and beyond, manifest the occurrence of sister chromatid exchanges (SCE), occurring both spontaneously and induced following exposure of the cells to the chemical aphidicolin (an inhibitor of DNA replication). Imaging SIMS emerges as a powerful investigative method for the study of chromosome structure and the elucidation of banding mechanisms, to assess the removal of proteins and DNA involved in chromosome preparation and in situ procedures, and in the study of a number of cytogenetic phenomena.

Aphidicolin↗
PubMed1995

Genomic organization and chromosomal localization of the gene TCF15 encoding the early mesodermal basic helix-loop-helix factor bHLH-EC2.

bHLH-EC2 is a recently characterized member of a growing family of basic helix-loop-helix transcription factors. This family includes bHLH factors such as twist, which appear to be primarily involved in early mesodermal differentiation, and bHLH factors such as TAL-1, which have been characterized through their association with chromosomal breakpoints associated with T-cell leukemias. To provide for studies aimed at understanding the genetic regulation of bHLH-EC2, we have characterized the organization of this gene and conducted preliminary studies of the transcriptional activity of the upstream promoter region. The mouse bHLH-EC2 gene was found to consist of two exons separated by a 5-kb intron, an organization pattern similar to the mouse twist gene. The transcription initiation site was identified by RNase protection assay and primer extension analysis. Linked promoter-reporter gene transfection experiments in cultured cells indicated that while the identified upstream sequence can function to promote transcription, it does not function in a cell-specific fashion. To investigate the possible association of bHLH-EC2 with hematological malignancy, the chromosomal location of this gene in the human was mapped by fluorescence in situ hybridization and assigned to chromosome band 20p13.

Animals↗
PubMed1995

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Amino Acid Sequence↗
PubMed1995

Genomic organization and chromosomal localization of the human and mouse genes encoding the alpha receptor component for ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) has recently been found to share receptor components with, and to be structurally related to, a family of broadly acting cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M. However, the CNTF receptor complex also includes a CNTF-specific component known as CNTF receptor alpha (CNTFR alpha). Here we describe the molecular cloning of the human and mouse genes encoding CNTFR. We report that the human and mouse genes have an identical intron-exon structure that correlates well with the domain structure of CNTFR alpha. That is, the signal peptide and the immunoglobulin-like domain are each encoded by single exons, the cytokine receptor-like domain is distributed among 4 exons, and the C-terminal glycosyl phosphatidylinositol recognition domain is encoded by the final coding exon. The position of the introns within the cytokine receptor-like domain corresponds to those found in other members of the cytokine receptor superfamily. Confirming a recent study using radiation hybrids, we have also mapped the human CNTFR gene to chromosome band 9p13 and the mouse gene to a syntenic region of chromosome 4.

Amino Acid Sequence↗
PubMed1995

Human glutamine: fructose-6-phosphate amidotransferase: characterization of mRNA and chromosomal assignment to 2p13.

It has been previously shown that some toxic effects of high concentrations of glucose are mediated by the hexosamine biosynthesis pathway and its rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). We have used the cloned human GFA cDNA to study the chromosomal localization of the gene and tissue distribution of mRNA. The human GFA gene is on chromosome 2, band p13 as determined by fluorescence in situ hybridization. An 8-kb species of GFA mRNA was detected in all rat tissues tested with relatively high expression in testis and smooth muscle; a unique 3-kb mRNA species was found only in testis.

Animals↗
PubMed1995

Abnormalities of chromosome band 11q23 and the MLL gene in pediatric myelomonocytic and monoblastic leukemias. Identification of the t(9;11) as an indicator of long survival.

PURPOSE AND METHODS: We reviewed the cytogenetic pattern of the malignant cells in 36 patients who were < 20 years of age and who had M4 and M5 leukemias, excluding M4Eo cases with inv(16). We performed fluorescence in situ hybridization (FISH) and molecular studies to determine the actual incidence of 11q23/MLL abnormalities in these patients. RESULTS: Eighteen patients had 11q23 translocations or insertions detected by cytogenetic analysis (15 cases) or by FISH (3 cases); 10 patients had t(9;11), all of whom had M5a. Eight patients had other 11q23 translocations or insertions not involving chromosome 9[t(11q23)] (four each had M4 or M5 leukemias). Eighteen cases with M4/M5 did not have 11q23 abnormalities. MLL rearrangements were found in all patients with translocations or insertions of 11q23 who were studied. Clinically, children with t(9;11) were indistinguishable from other patients with M4-M5 leukemias. In contrast, the t(11q23) group was characterized by extreme hyperleukocytosis, CNS disease, and skin involvement. Patients with the t(9;11) had a better outcome when compared with patients in the t(11q23) group (EFS +/- SE at 3 years, 56 +/- 17% versus 10 +/- 10%, p = 0.04), and to all the remaining children with M4-M5 leukemias (p = 0.04). CONCLUSIONS: The combination of cytogenetic, FISH, and molecular analysis provides a highly sensitive strategy for detection of 11q23/MLL gene rearrangements in childhood M4-M5 leukemias. Our more precise classification of these patients allows a more accurate correlation with outcome. The favorable prognostic significance of the t(9;11) should be confirmed in prospective studies including a larger number of children as well as adults.

Adolescent↗
PubMed1995

Antigenic and structural similarities between Mycobacterium tuberculosis 50- to 55-kilodalton and Mycobacterium bovis BCG 45- to 47-kilodalton antigens.

The relationship between Mycobacterium tuberculosis 50- to 55-kDa protein and Mycobacterium bovis BCG 45- to 47-kDa antigen was examined by using immunological and biochemical criteria. Reciprocal cross-reactivity with a rabbit polyclonal antiserum against the M. bovis BCG protein and with a monoclonal antibody raised against the M. tuberculosis antigen was observed. The epitope recognized by this antibody was apparently present only in proteins of M. tuberculosis and M. bovis BCG among the 11 mycobacterial species tested. The amino-terminal sequences and total amino acid contents of these proteins showed strong similarities. Both antigens are glycoproteins as assessed by binding of concanavalin A, labeling of carbohydrate moieties with biotin-hydrazide, and digestion of carbohydrates with jack bean alpha-D-mannosidase, which produced a reduction of the molecular weights of the proteins and totally eliminated concanavalin A binding. Both M. tuberculosis and M. bovis BCG proteins are secreted, since they were found mainly in the culture medium. Analysis of M. tuberculosis 50- to 55-kDa antigen by two-dimensional gel electrophoresis showed at least seven different components, as previously described for the M. bovis BCG antigen. Solid-phase immunoassays showed that the purified M. tuberculosis 50- to 55-kDa protein was recognized by serum specimens from 70% of individuals with pulmonary tuberculosis from a total of 77 Mexican patients examined.

Amino Acid Sequence↗
PubMed1995

Isolation of a cDNA clone encoding a KATP channel-like protein expressed in insulin-secreting cells, localization of the human gene to chromosome band 21q22.1, and linkage studies with NIDDM.

The metabolism of glucose in insulin-secreting cells leads to closure of ATP-sensitive K+ channels (KATP), an event that initiates the insulin secretory process. Defects in insulin secretion are a common feature of non-insulin-dependent diabetes mellitus (NIDDM), and the beta-cell KATP that couples metabolism and membrane potential is a candidate for contributing to the development of this clinically and genetically heterogeneous disorder. We screened a hamster insulinoma cDNA library by low-stringency hybridization with a probe coding for the G-protein-coupled inwardly rectifying K+ channel GIRK1/KGA and isolated clones encoding a protein, KATP-2, whose sequence is 90% similar to that of the recently described KATP-1, an ATP-sensitive K+ channel expressed in heart and other tissues. RNA blotting showed that KATP mRNA was present in insulin-secreting cells and brain but not in heart. To assess the contribution of KATP-2 to the development of NIDDM, the human KATP-2 gene (symbol KCNJ7) was isolated and mapped to chromosome band 21q22.1 by fluorescence in situ hybridization. A simple tandem repeat DNA polymorphism, D21S1255, was identified in the region of the KATP-2 gene, and linkage studies between this marker and NIDDM were carried out in a group of Mexican-American sib pairs with NIDDM. There was no evidence for linkage between D21S1255 and NIDDM, indicating that KATP-2 is not a major susceptibility gene in this population.

Adenosine Triphosphate↗
PubMed1995

Detection of 11q23/MLL rearrangements in infant leukemias with fluorescence in situ hybridization and molecular analysis.

Cytogenetic abnormalities of band 11q23 have been found in more than 50% of infant leukemias regardless of the phenotype. Using probes for the MLL gene at 11q23, MLL rearrangements have been identified in 70-80% of all infant leukemias including virtually all of the cases with 11q23 translocations, as well as cases with apparently normal karyotypes. We reviewed the chromosomal pattern of 26 cases of infant leukemias (12 ALL, 12 AML, two AUL). Eleven had 11q23 translocations, five had other abnormalities, and 10 had a normal karyotype. To determine whether 11q23/MLL rearrangements were present in the leukemia cells of patients with a normal karyotype, we performed FISH and molecular studies of eight of these patients who had adequate material. Three were found to have 11q23/MLL abnormalities, two of them detected by FISH; one ALL case had a t(11;19) (q23;p13.3), and one AML case had a t(11;19) (q23;p13.1). Retrospective review confirmed the presence of the t(11;19) in a small percentage of poor quality metaphase cells in both cases. A rearrangement of the MLL gene was detected by Southern blot analysis of leukemic cells from a third patient with ALL; one cell with a deletion of 11q23 was found on karyotypic review. Therefore, in our series the actual incidence of 11q23 abnormalities in infant leukemias was 54% (14/26): 67% in ALL (8/12) and 50% in AML (6/12). Our findings suggest that most infant leukemias with apparently normal karyotypes that have a molecular rearrangement of the MLL gene are undetected subtle translocations.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Aberrations↗