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G Alvarado-Urbina

Publications and source records attributed to G Alvarado-Urbina.

14 recordsLinked to original sources
PubMed1990

Probe amplifier system based on chimeric cycling oligonucleotides.

Amplification systems are required as part of DNA probe technology, since traditional non-amplified oligonucleotide hybridization using nonradioactive detection methods have detection limits of approximately 10(8) molecules. We present a probe amplifier technology suitable for use in large-scale automated clinical diagnostic systems. It is fast, sensitive and performs at a constant temperature. The system functions by allowing a single target molecule to act as a catalyst in converting a large number of probe molecules to a unique detectable form. We refer to this catalytic amplification process as the "cycling probe reaction." The basis of the system is an oligomer probe construction consisting of a DNA-RNA-DNA sequence.

Base Sequence↗
PubMed1989

Duplication of the phycocyanin operon in the unicellular cyanobacterium Anacystis nidulans R2.

Two phycocyanin (PC) operons, each containing alpha- and beta-subunit genes, have been isolated from the unicellular cyanobacterium Anacystis nidulans R2. Using oligodeoxyribonucleotide probes for the PC-coding regions, three PstI fragments were obtained and shown to contain the two operons, which are 2.7 kb apart, with a proposed gene order of 5'-(beta I-alpha I)-(beta II-alpha II)-3'. The nucleotide sequences of both alpha-subunit genes are identical, as are the beta-sequences and the 51-bp intergenic regions. However, significant nucleotide sequence differences are found in both the 5' and 3' untranslated regions of the two operons. Two mRNA species of 1.65 and 1.5 kb were detected in A. nidulans R2 RNA when probed with either the alpha-specific or the beta-specific probe. The results demonstrate the existence of two PC operons which are both transcriptionally active.

Base Sequence↗
PubMed1987

Phycocyanin alpha-subunit gene of Anacystis nidulans R2: cloning, nucleotide sequencing and expression in Escherichia coli.

The cloning and nucleotide sequence determination of the Anacystis nidulans R2 phycocyanin (PC) alpha-subunit gene are described. A 3.0-kb PstI fragment of Anacystis nidulans R2 genomic DNA cloned in plasmid pUC8 was found to hybridize with a heptadecameric oligodeoxynucleotide probe. Sequencing using synthetic primers revealed the presence of the PC alpha-subunit gene and the 3' proximal end of the beta-subunit gene. The alpha-gene is separated from the upstream beta-gene by a spacer length of 51 bp. The deduced amino acid (aa) sequence of the alpha-subunit protein is identical, except for 5 aa, to that of A. nidulans 6301 and is highly homologous (77%) to that reported for Agmenellum quadruplicatum PR6. The 16-kDa alpha-subunit protein, detected by immunoadsorption, was fortuitously expressed in Escherichia coli from the lacZ promoter of the cloning vehicle pUC8.

Amino Acid Sequence↗
PubMed1986

Rapid automated synthesis via diisopropyl phosphoramidite in situ activation. Chemical synthesis and cloning of a calmodulin gene.

A gene coding for a calmodulin was synthesized and cloned. The chemical synthesis of the gene, coding for 149 amino acids, was achieved by the enzymatic ligation of 61 chemically synthesized DNA fragments. The DNA fragments were synthesized using a solid support with a diisopropyl phosphoramidite intermediate and in situ activation. The automated standard cycle time was 10 min/addition. The synthesizer was designed and constructed from inexpensive, readily available parts and controlled by a Commodore 64 computer. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis.

Amino Acid Sequence↗
PubMed1985

Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis.

A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Site-specific modification of the lactose operator with acetylaminofluorene.

We have synthesized the tetradecamer GAGCXGATAACAAG containing a part of the sequence of the lactose operator. A guanine base in the sequence is replaced by the adduct of the carcinogen 2-acetylaminofluorene with guanine. Under the standard conditions of de-protection, the fluorene moiety is lost, leaving behind a guanine oxidation product. New conditions of de-protection have been developed which allow the isolation of an oligonucleotide containing the adduct of 2-aminofluorene with guanine. The presence of the aminofluorene adduct greatly increases retention on reverse phase chromatography and produces a unique pattern of sequencing bands.

2-Acetylaminofluorene↗
PubMed1981

Automated synthesis of gene fragments.

The DNA/RNA Synthesizer provides a complete and automated procedure for the synthesis of DNA sequences. Each base unit is added in a 30-minute cycle, permitting a tetradecamer to be constructed in 6 1/2 hours. The complete procedure is described, including a practical procedure for isolation and purification of the desired DNA sequence.

Automation↗
PubMed1979

Sensitive radioimmunoassay for somatostatin using N-[125I]-Tyr-somatostatin as labelled antigen.

A sensitive radioimmunoassay for somatostatin using N-[125I]-Tyr-somatostatin is described and compared with that using [125I]-Tyr1-somatostatin. The minimum detectable amount of somatostatin using N-[125I]-Tyr-somatostatin as tracer was 0.1 to 0.5 pg, which is approximately 10-fold lower than the lower detection limit of the RIA using [125I]-Tyr1-somatostatin. Moreover, it was found that the shelf-life of N[125I]Tyr-somatostatin was prolonged in comparison with labelled Tyr1-somatostatin. Human pancreatic and gastric extracts displayed immunological similarity to synthetic somatostatin tetradecapeptide.

Animals↗
PubMed1978

Enzymatic degradation of somatostatin by rat plasma and hypothalamus.

A highly sensitive and specific radioimmunoassay for somatostatin has been used to study inactivation of the neurohormone by plasma and hypothalamic peptidase(s). Specificity of the inactivation process was indicated by the absence of interference by addition of luteinizing hormone releasing hormone, thyrotropin-releasing hormone, oxytocin, or substance P. The inactivating ability of hypothalamic tissue and plasma was destroyed by heating and the protease inhibitor benzamidine prevented plasma activity, thus suggesting the enzymatic nature of the processes involved. The present data suggest that the inactivation of somatostatin by hypothalamus and plasma could be an important factor in the regulation of circulating somatostatin levels.

Animals↗
PubMed1977

The development of the hypothalamo-pituitary axis in the neonatal rat: hypothalamic somatostatin and pituitary and serum growth hormone concentrations.

Using a specific radioimmunoassay technique for somatostatin (GHRIH), we have studied the ontogenesis of hypothalamic GHRIH in relation to pituitary and serum GH concentrations in immature rats. Hypothalamic GHRIH concentrations rose from minimal levels of 4.5 +/- 0.2 pg/microgram protein (mean +/- SEM) at 2 days to peak concentrations of 40.6 +/- 4.1 pg/microgram protein at 28 days followed by a progressive decline toward 50 days (7.0 +/- 0.8 pg/microgram protein). Pituitary GH concentration attained peak prepuberal values of 203.5 +/- 22.8 ng/microgram protein at 16 days with a further marked rise after puberty. Serum GH concentration was elevated to 2 days (53.3 +/- 5.7 ng/ml) and declined progressively to 5.9 +/- 1.5 ng/ml at 13 days. There was a highly significant inverse correlation between hypothalamic GHRIH and serum GH concentrations (r = 0.743, P less than 0.005). These data indicate that the hypothalamic regulatory mechanism for pituitary GH release develops during the neonatal period of the rat and suggest that GHRIH may play an important physiological role in this process.

Aging↗