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D Valerio

Publications and source records attributed to D Valerio.

At least 109 records · Page 6Linked to original sources
PubMed1988

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.

Adenosine Deaminase↗
PubMed1987

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

Adenosine Deaminase↗
PubMed1986

One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA-SCID patient. In addition, wild-type sequences could be detected at the same positions, indicating a compound heterozygosity. Studies with ADA expression clones mutagenized in vitro showed that the mutation at position 304 is responsible for ADA inactivation.

Adenosine Deaminase↗
PubMed1985

Adenosine deaminase: characterization and expression of a gene with a remarkable promoter.

Cosmid clones containing the gene for human adenosine deaminase (ADA) were isolated. The gene is 32 kb long and split into 12 exons. The exact sizes and boundaries of the exon blocks including the transcription start sites were determined. The sequence upstream from this cap site lacks the TATA and CAAT boxes characteristic for eukaryotic promoters. Nevertheless, we have shown in a functional assay that a stretch of 135 bp immediately preceding the cap site has promoter activity. This 135-bp DNA fragment is extremely rich in G/C residues (82%). It contains three inverted repeats that allow the formation of cruciform structures, a 10-bp and a 16-bp direct repeat and five G/C-rich motifs (GGGCGGG) disposed in a strikingly symmetrical fashion. Some of these structural features were also found in the promoter region of other genes and we discuss their possible function. Knowledge of the exact positions of the intron-exon boundaries allowed us to propose models for abnormal RNA processing that occurs in previously investigated ADA-deficient cell lines.

Adenosine Deaminase↗
PubMed1985

Introduction of sequences encoding functional human adenosine deaminase into mouse cells using a retroviral shuttle system.

A retroviral packaging system was used to generate a murine virus carrying sequences encoding human adenosine deaminase (ADA). To this end, human ADA cDNA was inserted into the retroviral shuttle vector pZIP-NeoSV(X)1. This vector provides all of the cis-acting sequences necessary for the efficient packaging and transmission of the viral genome as well as a selectable gene for G418 resistance. Transfection of this recombinant plasmid into cells that provide essential virus products (psi-2 cells) yielded cell lines that stably produced virions carrying the coding sequence of human ADA. We have used these virions to infect NIH3T3 cells, which after 48 h synthesized catalytically active human ADA. Furthermore, G418-resistant cell lines were obtained from the virus-infected NIH3T3 cells that stably produced the human ADA enzyme.

Adenosine Deaminase↗
PubMed1984

Adenosine deaminase (ADA) deficiency in cells derived from humans with severe combined immunodeficiency is due to an aberration of the ADA protein.

In order to determine the molecular basis of adenosine deaminase (ADA) deficiency in cells derived from patients with severe combined immunodeficiency (SCID) disease, we used a human ADA cDNA clone (1) to analyse the organization and transcription of the ADA gene in both normal and ADA-SCID cells. In five lymphoblastoid ADA-SCID cell lines we could detect no deletions or rearrangements in the ADA gene and its flanking sequences. Furthermore, synthesis and processing of ADA mRNA appeared to be normal in the ADA-SCID cells, and ADA-specific mRNA from two ADA-SCID cells could be translated in vitro into a protein with the molecular weight of normal ADA; this protein, however, could hardly be precipitated with an ADA antiserum. The results indicate that in these two ADA-SCID cell lines, the lack of ADA activity is not due to transcriptional or translational defects, but to subtle changes in the configuration of the protein affecting both its enzymatic and immunological characteristics.

Adenosine Deaminase↗
PubMed1984

Cloning of human adenosine deaminase cDNA and expression in mouse cells.

A previously isolated partial cDNA sequence encoding human adenosine deaminase (ADA) was used to probe a cDNA library prepared from human cultured cell mRNA. Clones containing a combined overlapping length of 1462 bp were isolated and sequenced. One of these was found to include the entire ADA coding region. An open reading frame consisting of 363 codons was identified, predicting a polypeptide of Mr 40762. A mammalian expression plasmid was constructed, positioning the ADA coding sequence to be under transcriptional control of the mouse metallothionein promoter. Transfection of cultured mouse L-cells with this plasmid resulted in the acute expression of human ADA enzymatic activity, as assayed by isoelectric focusing.

Adenosine Deaminase↗
PubMed1983

Isolation of cDNA clones for human adenosine deaminase.

Clones encoding human adenosine deaminase (ADA) were isolated from a cDNA library made from the lymphoblastoid cell line MOLT-4. The isolation procedure was based on the selection of clones hybridizing with a radioactive probe complementary to an RNA preparation, which had been highly enriched in ADA-specific mRNA. The latter RNA preparation was obtained by size-fractionating MOLT-4 RNA and selecting fractions that were translatable into ADA. The assay for the presence of ADA in the in vitro translation products, was based on immunoprecipitation with a specific anti-ADA serum. The antiserum used was shown to precipitate a 42-kDal protein with the properties of ADA. Positive clones were further screened by means of hybrid-released in vitro translation assays. Two clones were obtained which were able to select mRNA that could be translated into a 42-kDal protein immunoprecipitable with the ADA-antiserum. By use of Southern blots containing DNA from somatic cell hybrids, one of these ADA cDNA clones was assigned to the human chromosome 20 known to contain the ADA gene.

Adenosine Deaminase↗
PubMed1983

[Ultrasonographic study in breast immersion].

100 breast symptomatic patients have been examined with ultrasound, using an automated water-path scanner (Senomatic 3D CGR), and commercial B-mode or real-time scanners. All the patients were also examined with xeromammography. Normal breast patterns, as well as benign and malignant breast lesions, are presented.

Adenofibroma↗
PubMed1982

An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.

Animals↗
PubMed1981

Single-layer anastomosis in the large bowel: ten years' experience.

Satisfactory experience with single-layer colorectal anastomosis during 1969-74 led to an analysis of the results of single-layer large bowel anastomosis during the subsequent years 1975-79. This technique has continued to be associated with a low incidence of anastomotic failure, but anastomotic integrity also depends on rigorous attitudes to bowel preparation, to bacterial contamination and to the avoidance of anastomosis when the risk of anastomotic failure is high.

Aged↗
PubMed1980

Large-bowel surgery, 1979: self-assessment.

Evidence of wide variability in the immediate results of large-bowel surgery stimulated self-assessment during 1979. The hazards of large-bowel surgery can usually be avoided by good bowel preparation, sound anastomotic technique, primary resection in large bowel emergencies, avoidance of anastomosis when hazardous, and antibiotic lavage for extant or potential peritoneal and wound contamination.

Adult↗