PubMed HealthSearch

Biomedical subjects

A Swaroop

Publications and source records attributed to A Swaroop.

At least 19 recordsLinked to original sources

A conserved retina-specific gene encodes a basic motif/leucine zipper domain.

Using subtraction cloning, we have isolated a human cDNA, AS321, which is expressed in retina and retinoblastoma cell lines but not in any other tissue or cell line tested. AS321 mRNA is detected in all cells of neural retina, with a high level of expression in photoreceptors. The polypeptide sequence deduced from the cDNA reveals consensus phosphorylation sites for protein kinase A and proline-directed protein kinase. Its C-terminal region contains a basic motif and a leucine zipper domain similar to the DNA binding proteins of the jun and fos oncoprotein family, and it shows a strong similarity to the product of an avian retroviral oncogene, v-maf. The gene for AS321 is conserved during evolution and is expressed in vertebrate retina. We propose to name the gene NRL (neural retina leucine zipper.

Amino Acid Sequence

Expressed sequence tags and chromosomal localization of cDNA clones from a subtracted retinal pigment epithelium library.

Expressed sequence tags (ESTs) provide useful molecular landmarks for physical mapping and identify the position of an expressed region in the genome. The use of subtracted cDNA libraries enriched for tissue-specific genes as a source of ESTs should reduce the repetitive isolation of constitutively expressed sequences. We report here the sequence tags from the 3'-end region of 58 new directionally cloned cDNAs from a subtracted human retinal pigment epithelium (RPE) cell line library. Eight of the cDNAs have been assigned to human chromosomes using PCR-based EST assays. Chromosomal mapping of subtracted RPE cDNA clones may also help in identifying candidate genes for inherited eye diseases.

Base Sequence

Neural retina-specific leucine zipper gene NRL (D14S46E) maps to human chromosome 14q11.1-q11.2.

The product of a neural retina-specific gene, NRL, belongs to the "leucine zipper" family of DNA-binding proteins and has a strong similarity to the v-maf oncogene product. The NRL gene maps to human chromosome 14 by Southern blot analysis of genomic DNA from a human-rodent somatic cell hybrid panel. In situ hybridization to metaphase chromosomes has further sublocalized the gene to the region 14q11.1-q11.2. D14S46E has now been assigned to the NRL gene. Because of its specific pattern of expression, NRL is a candidate gene for retinal diseases.

Animals

Differential expression of novel Gs alpha signal transduction protein cDNA species.

The Gs alpha guanine nucleotide-binding signal transduction protein is part of a heterotrimeric complex that is involved in the stimulation of adenylate cyclase upon activation of membrane receptors. We report the characterization of 16 Gs alpha cDNA clones isolated from the human adult retina and fetal eye libraries. Molecular heterogeneity in the 5'-region defines four novel Gs alpha cDNA species which are generated either by alternate splicing or by using alternative promoter. The novel exons upstream of exon 2 interrupt the highly conserved 'region A' in the Gs alpha polypeptide. Non-AUG codons in the novel 5'-exon can initiate translation of these Gs alpha species in vitro. Reverse transcription of total RNA coupled with polymerase chain reaction (RTPCR) using specific primers and in situ hybridization to mRNA in baboon tissue sections with a specific oligonucleotide probe show a high level of expression of these species in retina and brain but not in liver. Differential expression of alternatively spliced Gs alpha species suggests novel signal transducing pathways.

Aged

Sequence analysis, expression and chromosomal localization of a gene, isolated from a subtracted human retina cDNA library, that encodes an insulin-like growth factor binding protein (IGFBP2).

The metabolic functions of insulin-like growth factors (IGFs) I and II are modulated by a family of binding proteins which are present in biological fluids and are synthesized by a variety of cell types. A cDNA clone, isolated at random from a subtracted human retina library, has been identified to code for a novel IGF-binding protein (IGFBP2) by its sequence homology to the peptide sequence of IGF binding proteins purified from bovine MDBK and rat BRL-3A cells. The complete nucleotide sequence of the IGFBP2 cDNA is 1406 bp long, contains 66% G-Cs and an open reading frame of 328 amino acids with a putative signal or pro-peptide of 39 residues. The mature polypeptide of 289 amino acids has 18 cysteines, a putative ATP-binding site and an RGD tripeptide. The 1.4 kb IGFBP2 transcript is expressed in several human tissues including fetal eye and fetal brain, but not in the human lymphoblastoid cell line against which the retinal cDNA library was subtracted. In situ hybridization to sections of mouse retina localized the mRNA for IGFBP2 primarily in the outer nuclear layer of photoreceptors. Southern blot analysis of DNA from human x rodent and mouse x rodent somatic cell hybrids assigned the gene for IGFBP2 to human chromosome 2q33-qter and mouse chromosome 1 in a known conserved syntenic region.

Aged

Origin of mutations in two families with X-linked chronic granulomatous disease.

The most common X-linked recessive form of chronic granulomatous disease (X-CGD) is characterized by the absence of cytochrome b558 in neutrophils. In a rare variant form of X-CGD, cytochrome b558 is present but not functional. The gene (locus symbol CYBB) was localized to band Xp21 by studies of patients with small chromosome deletions. The gene was cloned based on its location and found to encode the 91-Kd subunit of the cytochrome b558 complex. Most female carriers for X-CGD can be identified by their X-inactivation mosaicism; on average 50% of their neutrophils express the mutant phenotype and fail to reduce nitroblue tetrazolium (NBT). In 2 of 4 families studied, the maternal grandmothers had normal NBT tests, suggesting either nonrandom X-inactivation or new mutations. Restriction fragment length polymorphism analysis using closely linked flanking markers or the NsiI polymorphism detected by the CYBB probe itself, allowed us to identify the X chromosome carrying the mutation as derived from a healthy NBT-positive maternal grandfather. The mothers of the affected boys must have received a paternal X chromosome carrying a new mutation, consistent with the maternal grandmothers' normal NBT tests. In all of eight potential carriers studied, the results of the NBT and DNA marker testing were in complete agreement. Prenatal diagnosis by DNA testing can be performed in early gestation obviating the need for fetal blood sampling.

DNA

Chromosomal localization and cDNA sequence of human ralB, a GTP binding protein.

The human ra1 proteins share more than 50% homology and some properties with the ras proteins. The RALB gene, a new member of the ra1 family, has been isolated, sequenced, and assigned to human chromosome 2 cen-q13. One other member of this family, RALA, has been previously mapped to the short arm of human chromosome 7.

Amino Acid Sequence

Molecular analysis of the cDNA for human SPARC/osteonectin/BM-40: sequence, expression, and localization of the gene to chromosome 5q31-q33.

Human cDNA clones encoding the extracellular calcium-binding, acidic glycoprotein known as SPARC, osteonectin, or BM-40 were isolated from a placental cDNA library. Two polyadenylated transcripts of 2.2 and 3.0 kb were detected in human tissues and cultured cells by Northern blot analysis, and cDNAs for both transcripts were characterized. The 2133-bp sequence of the more abundant (major) transcript contains an open reading frame for 303 amino acids. The deduced polypeptide has extensive amino acid sequence identity with mouse SPARC. The larger and minor 3.0-kb cDNA has an identical coding region but utilizes a downstream polyadenylation signal. Gene localization studies have revealed a single chromosomal site at 5q31-q33 by somatic cell hybrid analysis and in situ chromosomal hybridization. Furthermore, pulsed-field gel electrophoresis of human genomic DNA cleaved with different rare-cutting restriction enzymes and hybridized with SPARC cDNA probes revealed single or double fragments of less than 50 to about 150 kb. The evidence is consistent with a single locus for SPARC in humans. The gene was found to be differentially expressed in many human tissues and in an osteogenic sarcoma, but not in other transformed cells.

Amino Acid Sequence

Sequence analysis of the complete cDNA and encoded polypeptide for the Glued gene of Drosophila melanogaster.

The complete cDNA sequence for the Glued gene of wild-type Drosophila melanogaster contains an open reading frame encoding 1319 amino acids, which constitute the Glued polypeptide. The secondary predicted from the deduced sequence of the Glued polypeptide has extensive alpha-helical internal domains, which contain heptad-repeat sequences characteristic of an elongated coiled-coil conformation. There are striking sequence and conformation similarities between the Glued alpha-helical domains and those found in certain filamentous proteins from various organisms, particularly in muscle fibers and intermediate filaments. The possible role of the Glued polypeptide as an architectural filamentous component of Drosophila cells and tissues is discussed. Two of the five Glued exons are located in the 5' untranslated region of the cDNA. One of the introns interrupting the Glued open reading frame encodes at least two polyadenylylated transcripts, suggesting that other genes might map within the span of the Glued gene.

Amino Acid Sequence

Generation of H2O2 in brain mitochondria.

Generation of H2O2 by rat brain mitochondria using succinate and glycerol-1-phosphate as substrates has been demonstrated. Earlier workers were unable to detect this activity in sucrose-Tris buffer. We found that this was due to a lag in the expression of activity in sucrose medium. Using phosphate buffer (50 mM), good rates are now obtained. Generation of H2O2 by rat brain mitochondria required the presence of antimycin A and was dependent on the substrates succinate and glycerol-1-phosphate. Low rates were obtained with NAD+-linked substrates and none with choline, glutamate, and NADH. The Km and Vmax values for H2O2 generation were considerably lower than the corresponding values for the respective dehydrogenase activity, measured by dye reduction. Oxygen-radical scavengers inhibited H2O2 generation, suggesting oxygen radical involvement. Depletion of ubiquinone from mitochondria resulted in loss of H2O2 generation. Reconstitution of such depleted particles with ubiquinone restored the capacity to generate H2O2 in a concentration-dependent manner. Levels of H2O2 production were found to be maximal in cerebellum. Brain mitochondria from rabbit, hamster, mouse, and guinea pig also have the capacity to generate H2O2 on oxidation of glycerol-1-phosphate.

Animals

Molecular organization and expression of the genetic locus glued in Drosophila melanogaster.

The Glued locus of Drosophila melanogaster is genetically defined as the functional unit which is affected by the dominant Glued mutation Gl. Genomic DNA was cloned from the region of the Glued locus, at 70C2 on chromosome 3, by using a P element insertion in the region as a molecular marker. Three genes encoding polyadenylated transcripts were detected within a 30-kilobase span of the cloned DNA. The gene nearest the P element insertion site was identified as a Glued gene on the basis of alterations in its DNA and encoded transcript associated with the Gl mutation and with reversions of Gl which eliminate the dominant effect by inactivation of the mutant allele. Expression of the wild-type Gl+ gene is temporally regulated during development; the amount of the encoded transcript is highest in the embryonic stage, decreasing in the first and second larval instars, and then increasing in the third instar and pupal stages. There is a maternal contribution of the Gl+ transcript to the embryo, which probably accounts for the maternal lethal effect of Glued mutations on early development. In situ hybridizations of Gl+ DNA to RNA in tissue sections showed that the Gl+ transcript is present in virtually all tissues of the embryo, late larva, and pupa. The general distribution of this transcript is consistent with genetic evidence indicating that the Glued locus controls a generally essential cell function (P. J. Harte and D. R. Kankel, Genetics 101:477-501, 1982). Different Glued mutations produce distinct phenotypic effects, including adults with severe visual defects, larvae lacking imaginal discs, and early lethality. These diverse mutant phenotypes are discussed in terms of quantitative changes in the Glued function. Closely adjacent to Gl+ is another gene which is transcribed in a divergent direction and expressed coordinately with Gl+ throughout Drosophila development. It remains to be determined whether this gene is also involved with the Glued function.

Animals

Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria.

Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.

Animals

Molecular genetics of a transposon-induced dominant mutation in the Drosophila locus Glued.

The organization of the Drosophila locus Glued containing the dominant allele Gl was shown to differ from that of the normal locus by an insertion of a 9-kilobase-pair DNA segment near the 3' end of a transcribed region. The insertion causes the formation of a truncated polyadenylylated transcript of 5.1 kilobases instead of the normal 6.0 kilobases. The inserted DNA segment has the properties of a transposon and was identified by its corresponding restriction map as B104, which is a retrovirus-like transposon with direct terminal repeats. B104 appears to be oriented in Gl with the same polarity of transcription as Gl. The truncated Gl transcript terminates prematurely inside the 5' terminal repeat of B104, in the region of a putative polyadenylylation signal. We discuss the general implications of this finding for transposon- and retrovirus-induced mutagenesis and for the origin of dominant mutations.

Alleles