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PubMed2004

Xpro: database of eukaryotic protein-encoding genes.

Xpro is a relational database that contains all the eukaryotic protein-encoding DNA sequences contained in GenBank with associated data required for the analysis of eukaryotic gene architecture. In addition to the information found in the GenBank records, which includes properties such as sequence, position, length and description about introns, exons and protein-coding regions, Xpro provides annotations on the splice sites and intron phases. Furthermore, Xpro validates intron positions using alignment information between the record's sequence and EST sequences found in dbEST. In the process of validation, alternative splicing information is also obtained and can be found in the database. The intron-containing genes in the Xpro are also classified as experimental or predicted based on the intron position validation and specific keywords in the GenBank records that are present in predicted genes. An Entrez-like query system, which is familiar to most biologists, is provided for accessing the information present in the database system. A non-redundant set of Xpro database contents is also obtained by cross-referencing to the Swiss-Prot/TrEMBL and Pfam databases. The database currently contains information for 493,983 genes--351,918 intron- containing genes and 142,065 intron-less genes. Xpro is updated for each new GenBank release and is freely available via the internet at http://origin.bic. nus.edu.sg/xpro.

Alternative Splicing↗
PubMed2001

The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene.

Regions of the Streptomyces venezuelae ISP5230 chromosome flanking pabAB, an amino-deoxychorismate synthase gene needed for chloramphenicol (Cm) production, were examined for involvement in biosynthesis of the antibiotic. Three of four ORFs in the sequence downstream of pabAB resembled genes involved in the shikimate pathway. BLASTX searches of GenBank showed that the deduced amino acid sequences of ORF3 and ORF4 were similar to proteins encoded by monofunctional genes for chorismate mutase and prephenate dehydrogenase, respectively, while the sequence of the ORF5 product resembled deoxy-arabino-heptulosonate-7-phosphate (DAHP) synthase, the enzyme that initiates the shikimate pathway. A relationship to Cm biosynthesis was indicated by sequence similarities between the ORF6 product and membrane proteins associated with Cm export. BLASTX searches of GenBank for matches with the translated sequence of ORF1 in chromosomal DNA immediately upstream of pabAB did not detect products relevant to Cm biosynthesis. However, the presence of Cm biosynthesis genes in a 7.5 kb segment of the chromosome beyond ORF1 was inferred when conjugal transfer of the DNA into a blocked S. venezuelae mutant restored Cm production. Deletions in the 7.5 kb segment of the wild-type chromosome eliminated Cm production, confirming the presence of Cm biosynthesis genes in this region. Sequencing and analysis located five ORFs, one of which (ORF8) was deduced from BLAST searches of GenBank, and from characteristic motifs detected in alignments of its deduced amino acid sequence, to be a monomodular nonribosomal peptide synthetase. GenBank searches did not identify ORF7, but matched the translated sequences of ORFs 9, 10 and 11 with short-chain ketoreductases, the ATP-binding cassettes of ABC transporters, and coenzyme A ligases, respectively. As has been shown for ORF2, disrupting ORF3, ORF7, ORF8 or ORF9 blocked Cm production.

Amino Acid Sequence↗
PubMed2006

Analysis of the entire nucleotide sequence of hepatitis B virus genotype B in the Philippines reveals a new subgenotype of genotype B.

The entire nucleotide sequences were determined for hepatitis B virus (HBV) genotype B (HBV/B) genomes extracted from five patients in the Philippines and designated GenBank AB219426, AB219427, AB219428, AB219429 and AB219430. The serotype of the first four isolates was ayw and that of GenBank AB219430 was adw. Divergences of entire sequences were 1.0-2.0 % between the first four isolates and 3.8-4.2 % between these four and GenBank AB219430. Phylogenetic-tree analysis revealed that, worldwide, HBV/B comprises five subgenotypes: B1, B2, B3, B4 and the new Philippines group, designated B5. Divergences of the entire genome sequences between four isolates in subgenotype B5 and isolates from other countries (subgenotypes) were 4.4-4.8 % with Vietnam (B4), 2.9-3.5 % with Indonesia (B3), 4.7-5.1 % with China (B2) and 5.4-6.0 % with Japan (B1). Similarly, GenBank AB219430 showed the lowest divergences: 3.4 % with the isolate from Indonesia (B3), 5.0 % with Vietnam (B4), 5.4 % with China (B2) and 6.1 % with Japan (B1). This is the first report of entire nucleotide sequences of HBV/B from the Philippines and the results show that these sequences belong to a new subgenotype, B5. The present study identified that HBV/B isolates throughout the world are divided genetically into five subgenotypes, the relationships between geographical distances and the genetic distances of HBV/B being well-correlated.

Adult↗
PubMed2006

Identification and characterization of polymorphisms within the 5' flanking region, first exon and part of first intron of bovine GH gene.

The aim of the present study was to identify and characterize polymorphisms within the 5' flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental x Nelore (n = 30), Canchim x Nelore (n = 30) and Angus x Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648, AY662649 and AY662650, respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652, respectively). The allele sequences were compared with GenBank sequences (M57764, AF007750 and AH009106) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.

5' Flanking Region↗
PubMed2005

Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species.

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) chevalieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus (Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a < or = 1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a < or = 1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.

Aspergillus↗
PubMed2005

Approaching the taxonomic affiliation of unidentified sequences in public databases--an example from the mycorrhizal fungi.

BACKGROUND: During the last few years, DNA sequence analysis has become one of the primary means of taxonomic identification of species, particularly so for species that are minute or otherwise lack distinct, readily obtainable morphological characters. Although the number of sequences available for comparison in public databases such as GenBank increases exponentially, only a minuscule fraction of all organisms have been sequenced, leaving taxon sampling a momentous problem for sequence-based taxonomic identification. When querying GenBank with a set of unidentified sequences, a considerable proportion typically lack fully identified matches, forming an ever-mounting pile of sequences that the researcher will have to monitor manually in the hope that new, clarifying sequences have been submitted by other researchers. To alleviate these concerns, a project to automatically monitor select unidentified sequences in GenBank for taxonomic progress through repeated local BLAST searches was initiated. Mycorrhizal fungi--a field where species identification often is prohibitively complex--and the much used ITS locus were chosen as test bed. RESULTS: A Perl script package called emerencia is presented. On a regular basis, it downloads select sequences from GenBank, separates the identified sequences from those insufficiently identified, and performs BLAST searches between these two datasets, storing all results in an SQL database. On the accompanying web-service http://emerencia.math.chalmers.se, users can monitor the taxonomic progress of insufficiently identified sequences over time, either through active searches or by signing up for e-mail notification upon disclosure of better matches. Other search categories, such as listing all insufficiently identified sequences (and their present best fully identified matches) publication-wise, are also available. DISCUSSION: The ever-increasing use of DNA sequences for identification purposes largely falls back on the assumption that public sequence databases contain a thorough sampling of taxonomically well-annotated sequences. Taxonomy, held by some to be an old-fashioned trade, has accordingly never been more important. emerencia does not automate the taxonomic process, but it does allow researchers to focus their efforts elsewhere than countless manual BLAST runs and arduous sieving of BLAST hit lists. The emerencia system is available on an open source basis for local installation with any organism and gene group as targets.

Classification↗
PubMed2006

Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits.

BACKGROUND: The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL) for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10) near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR) gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine. RESULTS: Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4), which mapped to 126-128 cM on SSC10. Using the IMpRH7000rad and IMNpRH212000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07) and possibly ovulation rate (p = 0.102). Two SNP in AKR1C4 were significantly associated with nipple number (p </= 0.03) and another possibly associated with age at puberty (p = 0.09). CONCLUSION: AKR1C genotypes were associated with nipple number as well as possible effects on age at puberty and ovulation rate. The estimated effects of AKR1C genotypes on these traits suggest that the SNPs are in incomplete linkage disequilibrium with the causal mutations that affect reproductive traits in swine. Further investigations are necessary to identify these mutations and understand how these AKR1C genes affect these important reproductive traits. The nucleotide sequence data reported have been submitted to GenBank and assigned accession numbers [GenBank:DQ474064-DQ474068, GenBank:DQ494488-DQ494490 and GenBank:DQ487182-DQ487184].

Alcohol Oxidoreductases↗
PubMed2003

Comparative study on the expression of cytokine--receptor genes in normal and preeclamptic human placentas using DNA microarrays.

AIMS: To study the relationship between the expression levels of cytokine/receptor genes in placenta and the pathogenesis of pre-eclampsia. METHODS: The study was performed to compare the mRNA contents of cytokine (receptor) superfamily genes in placentas from 5 patients with pre-eclampsia and 5 strictly matched normal pregnancies. A complementary DNA microarray representing over 220 cytokine-associated genes was employed to complete the detection. RESULTS: It was shown that, among the 221 kinds of cytokine-associated genes, 162 of those including 22 interleukin/interleukin receptor genes presented with a difference of over two times in pre-eclamptic placentas compared to normal placentas. Most of the 22 interleukin/interleukin receptor genes were shown to be highly expressed in preeclamptic placenta, while the expression of IL-2 receptor (IL-2 R alpha, GenBank: X01057) gene in preeclamptic placenta was comparatively lower than that in normal placenta. Furthermore, some tumor necrosis factor (TNF)/receptor superfamily genes, including TNF (GenBank: X02910), TNF ligands (GenBank: U03398, U37518, AF053712, AF055872) and TNF receptors (GenBank: X60592, X63717, M83554, AF016266, AF016267, U81232) were also shown to be highly expressed in pre-eclamptic placenta. Besides interleukin and tumor necrosis factor (receptor) gene superfamily, the mRNA levels of another 39 cytokine and 15 cytokine receptor genes showed a two-fold difference between pre-eclamptic and normal placental tissues. Additionally, most of the genes were up-regulated in pre-eclamptic placenta. CONCLUSIONS: The up-regulation of cytokine-associated genes including interleukin and TNF (receptor) superfamily expression in placenta might be intensively related to the pathogenesis of pre-eclampsia.

Adult↗
PubMed2003

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii.

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.

Animals↗
PubMed2004

[Cloning of compaction-related gene Crg1 of mouse preimplantation embryos].

A total of 181 8-cell embryos and 241 8-cell compacted embryos were collected respectively from C57BL/6 mouse and their cDNA was synthesized by SMART-PCR. Then PCR productions after SSH were cloned into pUCm-T vector according to the size after isolated and purified. Choice the positive clones for sequencing after being confirmed with PCR. All fragment cloned were blastered matching in GenBank for homology analysis. On the base of this work, we select two ESTs (GenBank accession No.: BQ740263 and BQ740251), which were reverse complementary to each other, assemble into a cDNA fragment, which include a whole open read frame (ORF) and submitted to GenBank with an accession number, AY134859. The target fragment were amplified from cDNA of mouse compacted embryos using primers designed according to the ORF and cloned into pUCm-T vector and sequenced. It's confirmed that the gene, Crg1, was the same as AY134859. Crg1 were also blastered matching in GenBank for homology analysis and mapped by database analyses. RT-PCR analysis of Crg1 were done in series mouse embryos, mouse embryo stem cell and mouse embryo fibroblast. We analyzed the expression of Crg1 in 8-cell embryos and 8-cell compacted embryos using semi-quantity RT-PCR. Northern-blot analysis of Crg1 expression in some adult mouse tissues was done too. The results showed that the length of Crg1 is 810 bp. It include only one exon and code a 150 amino acid protein with a theoretical molecular weight of 17670.34 Dalton. The protein is similar to a protein encoded by a known gene, Stella. Crg1 was mapped to chromosome 14 by database analyses. RT-PCR analysis shows that Crg1 expressed in series mouse embryos (2-cell embryos, 4-cell embryos, 8-cell embryos, compacted embryos and blastocyst), and expressed little higher in compacted embryos. Crg1 also expressed in mouse embryo stem cell but not expressed in mouse embryo fibroblast. The gene only expressed weakly in adult mouse ovary, but not expressed in other adult mouse tissues (brain, spleen, heart, skeletal muscle, kidney, testis, epididymis, liver and lung). So, Crg1 may related to compaction in compacted embryos and maintaining cell's pluripotentiality.

Amino Acid Sequence↗
PubMed2004

[Preliminary screening of a reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

OBJECTIVE: To identify differentially expressed cDNA sequences for rat liver of prothrombotic state (PTS) by screening a reverse-subtracted cDNA library. METHODS: Forward-subtracted cDNA probes and reverse-subtracted cDNA probes were prepared by nested PCR amplification, which were labeled with HRP. Positive clones were selected by differential screening in which forward-subtracted and reverse-subtracted cDNA probes were separately hybridized with the membranes slot-blotted by plasmid DNAs amplified and isolated from the library. Inserts in the positive clones were submitted to DNA sequencing. Nucleic acid sequence homology search was performed against the GenBank DNA database (non-redundant, and non-mouse and non-human EST entries) using the Standard nucleotide-nucleotide BLAST [blastn] program via a network connection to the National Center for Biotechnology information. RESULTS: Five positive clones were found by differential screening. For 3 of the 5 sequences, known genes of high homology (Score>225 bits) in the GenBank DNA database were found. For the other 2 sequences, only genes of low homology (Score=56) or intermediate homology (Score=151) were found. CONCLUSION: Five cDNA sequences were found PTS related. 3 of them may represent the genes that are either identical or nearly identical to previously reported genes The other 2 sequences are most probably new sequences not existing in GenBank DNA database, which have been registered in the GenBank DNA database as new sequences.

Animals↗
PubMed2004

[Expression of some genes in Tamarix androssowii plant under NaHCO3 stress].

Differential display technique was used to study the gene expression of Tamarix androssowii treated with NaHCO(3) solution. Seventeen differentially expressed genes were obtained through Northern detection. The result of BLASTX analysis shows that two of these genes (GenBank accession number CD028574 and CD028579) are homologous to the F-box protein family member, which can regulate diverse cellular processes, including cell cycle transition, transcription regulation and signal transduction, by playing roles in Skp1p-cullin-F-box protein (SCF) complexes or non-SCF complexes. F-box protein plays a very important role in plant defence system. One gene (GenBank accession number CD028589) is highly homologous to the translation initiation factor eIF, which regulates the proteins translation and is an important determinant of sodium tolerance in yeast and plants. Additionally, two genes (GenBank accession numbers CD028584 and CD028586) that are homologous to secretory peroxidase and peroxidases were identified, peroxidases are familiar proteins expressed in plant under stress. Five new genes closely related to salt and alkali resistance were obtained, which were expressed quite differentially when treated by NaHCO(3) solution. Seven differentially expressed genes were obtained which have highly or some homologous to the known genes, their function in stress need to be further studied. These seventeen genes were accepted as novel cDNA sequences by GenBank and were given them new accession numbers, their accession numbers were CD028574- CD028581 and CD028583-CD028591.

Base Sequence↗
PubMed2005

[Cloning and analysis of LMW-GS genes from Triticum aestevum ssp. tibetanum Shao].

The full coding regions (open reading frame,ORF) of LMW-GS genes were amplified from Triticum aestevum ssp. tibetanum Shao accession AS908 by using the primer pairs W1227 and W1228,which were designed according to wheat LMW-GS conservative domains. The amplified DNA fragments were separated and recovered from agrose gel,subsequently ligated into pBluescript SK( +/-)-T vector, and then transformed into E. coil strain DH10B. Three positive clones were screened out and sequenced. Among which, Tibet1 (GenBank accession No:AY299457) and Tibet2 (GenBank accession No: AY299458) are two expressed LMW glutenin genes. Their coding regions are 1 041 bp and 901 bp, and encode two mature proteins with 326 and 281 amino acid residues, respectively. Tibet3 (GenBank accession No:AY299459) is a pseudogene due to that two stop codons are in its coding region. Squence multipule alignment analysis suggested that Tibet1, Tibet2 and Tibet3 have the higher similarity in their structure and quality functions with the known LMW-GS genes with GenBank accession No: AY214450, AJ293099 and AB062871 encoded by Glu-D3, Glu-A 3 and Glu-A3, respectively.

Amino Acid Sequence↗
PubMed2005

[Cloning and characterization of new genes of Schistosoma japonicum].

OBJECTIVE: To clone and characterize new genes of Schistosoma japonicum, Sj, and to provide efficient vaccine candidates. METHODS: Sj adult cDNA library was screened with rabbit sera raised against male worm soluble antigen. The inserted cDNA fragments from the positively selected clones were amplified with PCR and further sequenced, as well as characterized through internet NCBI GenBank software. RESULTS: Eleven positive clones were obtained and two were verified by GenBank as new, including a novel gene designated as Sj-P8 (GenBank accession No. AF517843) and a new partial cDNA of Sj myosin (GenBank accession No. AY770506). The two new genes encoded a transmembrane protein of 75 amino acids and a myosin protein fragment of 212 amino acids respectively. CONCLUSION: The newly obtained genes may provide useful information for the research on Sj vaccine.

Amino Acid Sequence↗
PubMed2005

[Cloning and characterization of the neutral trehalase gene in Metarhizium anisopliae CQMa102].

The partial fragment of the neutral trehalase (NTL) in Metarhizium anisopliae CQMa102 was amplified by the polymerase chain reaction (PCR) with primers designed according to the sequences of the NTL in GenBank. The amplified fragment was cloned and sequenced. Based on the known sequence of NTL gene, the 5' and 3' rapid amplification of cDNA ends (RACE) were used to amplify the 5' and 3'regions of the NTL cDNA, then the whole cDNA sequence of NTL gene in M. anisopliae CQMa102 was obtained by combining the above sequences and its whole genomic DNA was amplified by PCR. In order to obtain more regulatory information of the NTL, panhandle polymerase chain reaction strategy was used to amplify the 5' flanking sequences adjacent to the known sequence of the neutral trehalase gene. The sequence analysis shows that the DNA sequence is 3484bp in size, includes three introns, and the cDNA sequence is 2385 bp in size, encodes a protein of 737 amino acid residues with a calculated Mr of 83.1kD, in which there is a cyclic adenosine 3', 5'-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site, which is consistent with a regulatory enzyme. Comparison of this sequence with the NTL of M. anisopliae ME1 in GenBank (GenBank Accession: AJ298019, AJ298020) shows that the nucleotide homology is as high as 93%, and the amino acid homology comes up to 99%. Southern analysis indicated that NTL gene was present as a single copy in this M. anisopliae strain. A single transcript of approximately 2.5 kb was detected by Northern blot analysis. The NTL mRNA was present throughout spore germination in rich medium, but accumulated to its highest level at the early stage of exponential growth. Thereafter, the mRNA level declined at the late stage of exponential growth, but began to show an increase during the stationary phase of growth. A 982bp upstream sequence of NTL was amplified using panhandle PCR method, which contains one stress response element (STRE). The NTL nucleotide sequence and its amino acid sequence have been accessed by GenBank (Accession: AY557613, AY557612).

Animals↗
PubMed2002

Full-length cDNA cloning of human neuroglobin and tissue expression of rat neuroglobin.

Neuroglobin is a recently discovered respiratory, porphyrin-containing protein that is expressed in the brain of mouse and human. However, the full-length cDNA sequence and genomic organization of human neuroglobin have not been reported. In this paper, the full-length cDNA sequence of human neuroglobin was cloned following bioinformatic analysis and the rapid amplification of cDNA ends (RACE) technique. It was shown that the full-length cDNA sequence (GenBank Accession No. AF422796) of human neuroglobin is 1909 bp in size, and the genomic sequence is 8041 bp in size (GenBank Accession No. AF422797). To further study the characterization of this gene, the coding region of rat neuroglobin (GenBank Accession No. AF333245) was cloned by using degeneracy PCR. The result showed high conservation among human, rat, and mouse neuroglobin. Furthermore, it was demonstrated that NGB was extensively expressed in rat brain by using in situ hybridization and the immunohistochemical technique. Transcription of NGB mRNA was shown to be widely distributed throughout the adult rat brain, including cerebral cortex, hippocampus, thalamus, hypothalamus, olfactory bulb, and cerebellum. NGB protein immunoreactive cells were also widely distributed throughout normal adult rat brain, including cerebral cortex, hippocampus, thalamus, hypothalamus, pons, and cerebellum. It could be seen that the NGB-immunopositive signals were in the cytoplasma and processes of the neuron. These data strongly support the notion that neuroglobin is a highly conserved gene in evolution and is very important in the nervous system, possibly related to the oxygen supply of the neuron.

Aging↗
PubMed1999

Molecular characterization of a MSRV-like sequence identified by RDA from monozygotic twin pairs discordant for schizophrenia.

Retroviral-related amplicons were used in modified RDA to identify four sequences from affected members of three pairs of monozygotic twins discordant for schizophrenia. One sequence (schizophrenia associated retrovirus, SZRV-1, GenBank Accession No. AF135487) is characterized here. It is similar to two known sequences of retroviral origin: multiple sclerosis-associated retrovirus, MSRV (GenBank Accession No. AF009668), and ERV-9 (GenBank Accession No. S77575). It is present in multiple copies in the human genome and has been localized to six different chromosomal sites. A zooblot shows that this multicopy sequence is predominant in the primate lineage and present in rhesus monkeys and humans. SZRV-1 is expressed as a 9-kb RNA band in the placenta. This could offer support to the hypothesis that retroviral sequences transposing during fetal growth may alter neurodevelopmental genes and cause diseases, although its direct involvement in the causation of schizophrenia remains to be established.

Blotting, Southern↗
PubMed2000

Preparation of recombinant bovine, porcine, and porcine W4R/R5K leptins and comparison of their activity and immunoreactivity with ovine, chicken, and human leptins.

Recombinant ovine Ala-leptin (GenBank Accession No. U84247, of ovine leptin), previously prepared in our laboratory in prokaryotic expression plasmid pMON3401, was mutated using a mutagenesis kit to prepare plasmids encoding for bovine (GenBank Accession No. U50365) and porcine (GenBank Accession No. U59894) leptins and for porcine leptin analogue W4R/R5K. Escherichia coli cells transformed with these plasmids overexpressed large amounts of these proteins upon induction with nalidixic acid. The expressed proteins, found in inclusion bodies, were refolded and purified to homogeneity using subsequently anion- and cation-exchange chromatography. All three purified proteins showed a single band of the expected molecular mass of 16 kDa in SDS-PAGE in the presence of reducing agent and were composed of 90-100% monomers. Proper refolding was evidenced by comparing their CD spectra to those of previously prepared chicken and ovine leptins and to commercially available human leptin. The amino acid content of the purified proteins closely resembled the predicted composition. The biological activity of bovine leptin, porcine leptin, and porcine leptin analogue W4R/R5K was evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor. All three proteins, as well as ovine and chicken leptins, but not human leptin, exhibited a very high degree of cross-immunoreactivity against antiserum raised against ovine leptin in rabbits. In contrast, none or very low cross-immunoreactivity was observed against antiserum raised against ovine leptin in goats.

Amino Acid Sequence↗