Biomedical subjects

M B Soares

Publications and source records attributed to M B Soares.

At least 55 records · Page 3Linked to original sources

PubMed1996

Discovering distinct genes represented in 29,570 clones from infant brain cDNA libraries by applying sequencing by hybridization methodology.

To discover all distinct human genes and to determine their patterns of expression across different cell types, developmental stages, and physiological conditions, a procedure is needed for fast, mutual comparison of hundreds of thousands (and perhaps millions) of clones from cDNA libraries, as well as their comparison against data bases of sequenced DNA. In a pilot study, 29,570 clones in duplicate from both original and normalized, directional, infant brain cDNA libraries were hybridized with 107-215 heptamer oligonucleotide probes to obtain oligonucleotide sequence signatures (OSSs). The OSSs were compared and clustered based on mutual similarity into 16,741 clusters, each corresponding to a distinct cDNA. A number of distinct cDNAs were successfully recognized by matching their 107-probe OSSs against GenBank entries, indicating the possibility of sequence recognition with only a few hundred randomly chosen oligomers.

Animals↗

PubMed1996

Normalization and subtraction: two approaches to facilitate gene discovery.

Large-scale sequencing of cDNAs randomly picked from libraries has proven to be a very powerful approach to discover (putatively) expressed sequences that, in turn, once mapped, may greatly expedite the process involved in the identification and cloning of human disease genes. However, the integrity of the data and the pace at which novel sequences can be identified depends to a great extent on the cDNA libraries that are used. Because altogether, in a typical cell, the mRNAs of the prevalent and intermediate frequency classes comprise as much as 50-65% of the total mRNA mass, but represent no more than 1000-2000 different mRNAs, redundant identification of mRNAs of these two frequency classes is destined to become overwhelming relatively early in any such random gene discovery programs, thus seriously compromising their cost-effectiveness. With the goal of facilitating such efforts, previously we developed a method to construct directionally cloned normalized cDNA libraries and applied it to generate infant brain (INIB) and fetal liver/spleen (INFLS) libraries, from which a total of 45,192 and 86,088 expressed sequence tags, respectively, have been derived. While improving the representation of the longest cDNAs in our libraries, we developed three additional methods to normalize cDNA libraries and generated over 35 libraries, most of which have been contributed to our integrated Molecular Analysis of Genomes and Their Expression (IMAGE) Consortium and thus distributed widely and used for sequencing and mapping. In an attempt to facilitate the process of gene discovery further, we have also developed a subtractive hybridization approach designed specifically to eliminate (or reduce significantly the representation of) large pools of arrayed and (mostly) sequenced clones from normalized libraries yet to be (or just partly) surveyed. Here we present a detailed description and a comparative analysis of four methods that we developed and used to generate normalize cDNA libraries from human (15), mouse (3), rat (2), as well as the parasite Schistosoma mansoni (1). In addition, we describe the construction and preliminary characterization of a subtracted liver/spleen library (INFLS-SI) that resulted from the elimination (or reduction of representation) of -5000 INFLS-IMAGE clones from the INFLS library.

Adult↗

PubMed1995

Sequence characterization and genetic mapping of the human VSNL1 gene, a homologue of the rat visinin-like peptide RNVP1.

In the course of isolation and sequence analysis of microsatellite repeat containing human cDNAs, we have isolated the human homologue of the rat visinin-like peptide gene. The human gene shows a high degree of conservation at both the amino acid and the DNA sequence level. The (CA)n microsatellite repeat embedded in the 3' untranslated region of the gene is conserved between rat and human, along with the flanking DNA sequences. We have mapped the VSNL1 gene to the short arm of chromosome 2.

Amino Acid Sequence↗

PubMed1995

Identification of new Schistosoma mansoni genes by the EST strategy using a directional cDNA library.

A directional size-selected cDNA library constructed from Schistosoma mansoni (Sm) adult worm RNA was used for the generation of expressed sequence tags (EST). From one or both ends of 429 distinct cDNA clones 607 EST were obtained. Of these, only 16% were previously known Sm genes. More than 22% of the clones had matches with entries for other organisms in the databases. These new Sm genes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, membrane, nuclear and secretory proteins, and proteins with other functions. Almost 33% of the clones had no significant database matches and thus potentially represent Sm-specific genes. Among the latter, several clones, as judged by their redundancy in the library, appear to represent abundant transcripts. The data, taken as a whole, more than double the number of Sm genes identified by nucleotide sequencing and indicate the potential value of the adoption of genome sequencing strategies for the rapid increase in knowledge of complex disease-causing organisms.

Animals↗

PubMed1995

Gene-based sequence-tagged-sites (STSs) as the basis for a human gene map.

Using our data set of 3,143 single pass sequences from human brain cDNA libraries, we have developed a strategy in which gene-based sequence-tagged-sites (STSs), derived from 3'untranslated regions of human cDNAs, are rapidly assigned to megabase-insert yeast artificial chromosomes and somatic cell hybrids to generate regional gene mapping data. Employing this approach, we have mapped 318 cDNAs, representing 308 human genes. Ninety-two of these mapped to regions implicated in human genetic diseases, identifying them as candidate genes. Extension of this strategy has the potential to result in virtually every human gene having, at its 3' end, its own associated STS, with each STS in turn specifying both a corresponding genomic clone and a specific regional location in the genome.

Animals↗

PubMed1994

Construction and characterization of a normalized cDNA library.

We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

Base Sequence↗

PubMed1994

Selection of cDNAs using chromosome-specific genomic clones: application to human chromosome 13.

We have developed a general method for en masse isolation of cDNAs present in a normalized library by hybridization to arrayed chromosome-specific phage lambda clones; we have used this approach to initiate exon-mapping of human chromosome 13. An advantage of the simultaneous isolation of cDNA/lambda pairs is that it allows cytogenetic assignment of a bona fide genomic clone by in situ hybridization, which also verifies that the corresponding cDNA or a homologous expressed sequence resides on chromosome 13. This information is enriched by partial sequencing of a selected cDNA from both ends. The sequence of the 3' noncoding region provides an 'identifier' that is used to develop STSs, while the sequence from the 5' end, often corresponding to a coding region, is used for homology searches in databases that occasionally reveal gene functions.

Amino Acid Sequence↗

PubMed1993

The Trypanosoma cruzi ribosomal RNA-encoding gene: analysis of promoter and upstream intergenic spacer sequences.

The transcription start point (tsp) of the ribosomal RNA(rRNA)-encoding gene of Trypanosoma cruzi was mapped at 1550 bp upstream from the 18S rRNA coding sequence. The + 1 nucleotide (tsp) was determined to be a guanosine. As described for other eukaryotes, no consensus sequence was found when the putative promoter sequence (-200 to + 50) was compared with that described for Trypanosoma brucei and Crithidia fasciculata. However, a repeated element was found in the upstream intergenic spacer sequence (IGS) of T. cruzi. Motifs, present in this element, exhibit significant homology to the T. cruzi promoter sequence. Furthermore, the same motifs could be found, in a similar sequence organization, within the T. brucei promoter region. Therefore, the data described in this paper strongly indicate that the IGS rDNA (DNA coding for rRNA) organization in trypanosomatids appears similar to that found in higher eukaryotes.

Animals↗

PubMed1993

A microsatellite genetic linkage map of human chromosome 13.

We have characterized 21 polymorphic (CA)n microsatellites for the development of a genetic map of chromosome 13. Fifteen markers were isolated from a flow-sorted chromosome 13 library, four CA repeats were derived from NotI-containing cosmid clones, and two polymorphic markers were described previously (J. L. Weber, A. E. Kwitek, and P. E. May, 1990, Nucleic Acids Res. 18: 4638; L. Warnich, I. Groenwald, L. Laubscher, and A. E. Retief, 1991, Am. J. Hum. Genet. 49(Suppl.): 372 (Abstract)). Regional localization for all of the markers was performed by amplification of DNA from five somatic cell hybrids containing different deletions of chromosome 13. Genetic markers were shown to be distributed throughout 6 of the 11 resolvable chromosomal subregions. Using data from nine families provided by the Centre d'Etude du Polymorphisme Humain (CEPH), a framework map of 12 of these 21 markers was developed. Six of the 12 markers form three pairs, with each two members of a pair being tightly linked, such that nine systems of markers can be distinguished. The average heterozygosity of these 12 markers is 0.75. The total length of the sex-averaged map is 65.4 cM (Kosambi), with an average distance of 8.2 cM between systems of markers (eight intervals). Seven remaining markers were placed provisionally into the framework map.

Alleles↗

PubMed1993

Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library.

A human infant brain cDNA library, made specifically for production of expressed sequence tags (ESTs) was evaluated by partial sequencing of over 1,600 clones. Advantages of this library, constructed for EST sequencing, include the use of directional cloning, size selection, very low numbers of mitochondrial and ribosomal transcripts, short polyA tails, few non-recombinants and a broad representation of transcripts. 37% of the clones were identified, based on matches to over 320 different genes in the public databases. Of these, two proteins similar to the Alzheimer's disease amyloid precursor protein were identified.

Amino Acid Sequence↗

PubMed1992

Ribosomal DNA restriction analysis and synthetic oligonucleotide probing in the identification of genera of lower trypanosomatids.

Fifty-four species or isolates of insect trypanosomatids were examined for the presence of selected restriction enzyme sites in the small (SSU) and large (LSU) rRNA coding units of ribosomal genes. In the SSU, sites for Eco RI, Bgl II, Pst I, and Hind III were found to occur at the same location for all species examined, thus displaying a universal distribution among trypanosomatids. In the LSU, a site for Bgl II in the 24S-alpha sequence and sites for Hind III and Pst I in the 24S-beta sequence were found in all species examined. In contrast, a site for Pvu II in the SSU exhibited a genus-related distribution, being present in Crithidia and Herpetomonas but absent in Phytomonas. A site for Hind III in the 24S-alpha sequence of the LSU also exhibited genus-restricted distribution. The site was present in Crithidia but absent in Phytomonas and Herpetomonas. These findings were confirmed by dot hybridization with a synthetic oligonucleotide complementary to the 18S rRNA sequence containing the Pvu II site. Results point to the usefulness of restriction markers as diagnostic tools for distinguishing the lower trypanosomatid genera Crithidia, Herpetomonas, and Phytomonas at the same time revealing a marked complexity within the genus Leptomonas.

Amidohydrolases↗

PubMed1990

Restriction fragment length polymorphisms in the ribosomal gene spacers of Trypanosoma cruzi and Trypanosoma conorhini.

The ribosomal RNA genes of two species of Trypanosoma, Trypanosoma cruzi, the etiological agent of Chagas' disease, and Trypanosoma conorhini, a non-pathogenic rodent trypanosome, were cloned and partially characterized. The physical maps derived for their rRNA genes were similar throughout the region that encompasses the SSU-and LSU-rRNA coding sequences. However, the non-transcribed spacer DNA of both T. cruzi and T. conorhini was found to be polymorphic for several restriction enzyme sites. We show that strains of T. cruzi can be typed according to the characteristic restriction fragment length polymorphism of their NTS DNAs.

Animals↗

PubMed1990

[Benzodiazepines: patterns of use, tolerance and dependence].

The authors review recent studies on benzodiazepine, the most largely used drug for insomnia and anxiety. In this paper are summarized: the development, patterns of use and abuse, mechanism of action, development of differential tolerance to its many effects, and the phenomena of withdrawal and dependence on the benzodiazepines.

Anti-Anxiety Agents↗

PubMed1988

Expression of the insulin-like growth factor II gene in the choroid plexus and the leptomeninges of the adult rat central nervous system.

The rat insulin-like growth factor II gene, encoding a fetal somatomedin, expresses a multitranscript family in embryonic/fetal tissues and in the adult brain and spinal cord. By performing in situ hybridization on tissue sections of adult brain and spinal cord, we have found that these transcripts are not expressed in neural or glial cells but are expressed in the epithelium of the choroid plexus of each cerebral ventricle and in the leptomeninges. We propose that the choroidal epithelial cells synthesize and secrete insulin-like growth factor II into the cerebrospinal fluid.

Animals↗

PubMed1986

Rat insulin-like growth factor II gene. A single gene with two promoters expressing a multitranscript family.

We have characterized the single-copy rat gene encoding the protein precursor of insulin-like growth factor II (pre-pro-rIGF-II) that is located downstream from and in the same transcriptional orientation as the homologous insulin II gene (5'-insulin-IGF-II-3'). This gene consists of at least three coding exons and utilizes two promoters that generate alternate 5' non-coding exons. Multiple transcripts from both promoters appear primarily in fetal or neonatal tissues (in all of the developmental stages and tissues that we have examined), but they are extremely rare or undetectable in adult tissues, with the exception of the brain and the spinal cord. These transcripts, which exhibit characteristic developmental profiles in various tissues, differ both in the presence of one of the alternate 5' non-coding exons and in the length of their fourth exon. The possible occurrence of differential splicing or differential polyadenylation (or both) in this region is discussed.

Animals↗

PubMed1985

Developmental and tissue-specific expression of a family of transcripts related to rat insulin-like growth factor II mRNA.

We have constructed a cDNA library from the mRNA of a rat liver cell line (BRL-3A) and characterized cDNA clones encoding the protein precursor of the rat insulin-like growth factor II (pre-pro-rIGF-II). This precursor, inferred from the nucleotide sequence, consists of a signal peptide, the rIGF-II sequence, and a trailer polypeptide of unknown significance. The characterized cDNA sequence (1016 nt) is part of a 3.4 kb mRNA species. Northern analysis reveals that a probe containing the extreme 5' noncoding region hybridizes to a second RNA (1.6 kb), while a probe corresponding to the 5' noncoding region proximal to the coding region hybridizes to two other RNA species (1.75 and 1.1 kb). All four RNAs are differentially expressed in all of the neonatal tissues that were examined, while the 3.4 kb pre-pro-rIGF-II mRNA and the 1.1 kb transcript are absent from adult tissues.

Amino Acid Sequence↗

PubMed1985

Rat LINE1: the origin and evolution of a family of long interspersed middle repetitive DNA elements.

We present approximately 7.0 kb of composite DNA sequence of a long interspersed middle repetitive element (LINE1) present in high copy number in the rat genome. The family of these repeats, which includes transcribing members, is the rat homologue of the mouse MIF-Bam-R and human Kpn I LINEs. Sequence alignments between specimens from these three species define the length of a putative unidentified open reading frame, and document extensive recombination events that, in conjunction with retroposition, have generated this large family of pseudogenes and pseudogene fragments. Comparative mapping of truncated elements indicates that a specific endonucleolytic activity might be involved in illegitimate (nonhomologous) recombination events. Sequence divergence analyses provide insights into the origin and molecular evolution of these elements.

Amino Acid Sequence↗

PubMed1985

RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon.

Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome I but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes. DNA sequence comparisons of the gene-flanking regions in rats and mice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.

Animals↗