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PubMed2004

Molecular cloning and characterization of rat karyopherin alpha 1 gene: structure and expression.

Dopamine denervation in the striata of patients with Parkinson's disease (PD) leads to changes in neural plasticity. However, the mechanisms leading to the changes are still poorly understood. In an effort to study the molecular events in the denervated striatum, we identified and cloned rat karyopherin alpha 1 (KPNA1), a member of the importin/karyopherin alpha (KPNA) family. DNA sequence analysis revealed that the full-length cDNA, encoding rat KPNA1, was 4975 bp with a short 5'-untranslated region (UTR) of 70 bp, a putative coding sequence of 1617 bp, and an unusually long 3'-UTR of 3266 bp. The gene shared a high degree of similarity with its mouse and human homologs at both cDNA and protein levels. By computational analysis of its genomic sequence, the transcription unit was shown to span a 44-kb region and consist of 13 exons varying in size from 89 (6th exon) to 3454 bp (13th exon), and 12 introns varying in size from 0.3 to 8.9 kb. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that KPNA1 transcript existed in various adult tissues. Both Northern blot and semi-quantitative RT-PCR analysis showed that the expression level of KPNA1 mRNA was altered in the denervated striatum post-lesion in a time-dependent manner, reaching the maximum at 2 weeks post-lesion. Our results suggest involvement of KPNA1 in the striatal responses to denervation following 6-hydroxydopamine (6-OHDA)-induced lesion.

3' Flanking Region↗
PubMed1995

Molecular and immunological characterization of a 64-kDa protein of Actinobacillus actinomycetemcomitans.

The 64-kDa protein to which about half the sera from patients with localized juvenile periodontitis and rapidly progressive periodontitis reacted strongly was purified from Actinobacillus actinomycetemcomitans Y4. Determination of the N-terminal sequence of the protein revealed that it was a GroEL-like protein. The DNA fragment containing the groEL gene of A. actinomycetemcomitans was amplified by polymerase chain reaction, and the groESL operon was cloned by using colony hybridization with the amplified fragment from A. actinomycetemcomitans chromosomal DNA. Sequence analysis revealed that structures of the operon and its products were typical in gram-negative bacteria. Rabbit polyclonal antibodies to the 64-kDa protein cross-reacted with approximately 65-kDa proteins of Haemophilus aphrophilus, Haemophilus influenzae, Haemophilus paraphrophilus, Escherichia coli and Eikenella corrodens but not with any cellular proteins of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum. It is possible that antibodies reactive to the 64-kDa protein in periodontitis patients are induced by the cross-reactivity with the hsp60 proteins of other bacteria.

Aggregatibacter actinomycetemcomitans↗
PubMed1998

Gene expression induced by physical impedance in maize roots.

Two cDNA clones, pIIG1 and pIIG2, corresponding to mRNAs that accumulate in maize root tips subjected to 10 min of physical impedance, were isolated by differential screening of a cDNA library. The deduced proteins, based on DNA sequence analysis, have molecular masses of 13 and 23 kDa for pIIG1 and pIIG2, respectively. pIIG1 showed 97% similarity at the nucleic acid level to a maize root cortical cell delineating protein (pZRP3) and was also similar to some bimodular proteins that are developmentally or stress regulated in other plant species. In situ localization of pIIG1 showed some expression in cortical cells of control maize roots; however, after a 10 min physical impedance treatment, pIIG1 accumulation increased greatly in cortical cells and extended to include the procambial region. pIIG2 did not show sequence similarity with any identified gene of known function, but a bipartite nuclear targeting sequence occurs in its deduced amino acid sequence which indicates it may function in the nucleus. Thus, rapid accumulation of specific mRNAs occurs in maize roots in response to impedance stress, and these mRNAs may be responsible for some responses of the roots to physical impedance.

Amino Acid Sequence↗
PubMed2000

Esr genes show different levels of expression in the same region of maize endosperm.

Esr genes share high homology among each other, code for small hydrophilic proteins, and are expressed in a restricted region of maize endosperm surrounding the embryo. We show here that not only Esr2 but also Esr1 and Esr3 are expressed in maize, and that the relative contribution of Esr1, Esr2 and Esr3 to total Esr mRNA is 17%, 55% and 28%, respectively. DNA sequence analysis of putative promoter fragments ranging from 0.53 kb to 3.54 kb revealed the presence of retrotransposons related to the Zeon and Cinful families in the distal parts of the promoters. The proximal parts show high homology that extended over 504bp between Esr2 and Esr3, and 265bp between Esr1 and the other two genes. The most conspicuous potential cis element is a fully conserved tandem repeat of the sequence CTACACCA close to the respective open reading frames (ORFs). By the analysis of transgenic maize plants carrying promoter-Gus fusions, it was shown that all three cloned upstream fragments contain functional promoters, that the spatial activity of all three Esr promoters is identical, and that the cis element(s) responsible for the expression in the embryo surrounding region reside in the 265 bp upstream of the respective ORFs.

Base Sequence↗
PubMed1996

Molecular dissection of corticosteroid action in the rat hippocampus. Application of the differential display techniques.

Both adrenal steroids and glutamate are crucial for hippocampal cell viability. In order to identify adrenal steroid- and glutamate-responsive genes controlling hippocampal cell viability, we have used the PCR-based differential display method. We have described the characteristics of this technique and how it can be automated. Using differential display, we have identified a number of rat hippocampal genes of which the expression is affected by a combination of the glutamate analog kainic acid and adrenalectomy. Administration of kainic acid or removal of the adrenals alone gave a limited number of differentially displayed genes. Therefore, our results indicate that the main mode of corticosteroid receptor-controlled gene expression in the hippocampus is interaction with other transcription factors (e.g., CREB, AP-1) and not by binding to hormone-responsive elements of corticosterone-specific genes. Characterization by multiplex PCR experiments of a differentially displayed fragment of which the expression is increased by the combination of kainic acid and adrenalectomy confirmed our differential display results. Further characterization by DNA sequence analysis of the corresponding full-length cDNA clone revealed a gene product with 91.4% sequence identity with the mouse transcription factor KROX-20, suggesting that we have cloned the rat homolog. This finding suggests a role of KROX-20 in corticosteroid- and kainic acid-controlled hippocampal plasticity.

Adrenalectomy↗
PubMed2001

Cloning, expression and chromosomal location of NKX6B TO 10Q26, a region frequently deleted in brain tumors.

Nkx6-2 (former Gtx) is a murine-homeobox-containing gene localized distally on Chromosome (Chr) 7. Analysis of the expression pattern, together with DNA binding assays, suggests that this gene product might be important for differentiated oligodendrocyte function and in the regulation of myelin gene expression. We now report on the cloning and characterization of the human homolog (NKX6B). DNA sequence analysis of an 11-kb genomic fragment revealed that the complete human gene spans 1.2 kb and is composed of three exons. NKX6B is predicted to encode a polypeptide of 277 amino acids with 97% identity to mouse Nkx6-2. Northern blot experiments showed that NKX6B expression is tightly controlled in a tissue-specific fashion with the highest site of expression being the brain. Finally, using STS content mapping and RH analyis, we demonstrated that NKX6B maps to the 10q26, a region where frequent loss of heterozygosity has been observed in various malignant brain tumors. These results may implicate NKX6B as a candidate tumor suppressor gene for brain tumors, particularly for oligodendrogliomas.

Amino Acid Sequence↗
PubMed2005

[Ribosomal protein genes highly expressed in swamp eel gonads].

8 cDNA clones have been isolated from a cDNA library prepared from swamp eel testies by macroarray. DNA sequence analysis and database search showed that they encode 8 proteins which are highly homologous to 40S ribosomal proteins S4,S9,S16,S17,S20 and 60S riobosomal proteins L7, L18a,L29. Phylogenetic trees (ML) based on ribosomal protein genes from swamp eel and other organisms has been reconstructed, which showed that ribosomal protein genes were highly conserved during evolution. These results suggested that ribosomal protein genes as house keeping genes may play roles in developmental regulation such as sexual differentiation and can also be used as markers for the study of molecular evolution.

Amino Acid Sequence↗
PubMed1996

Cloning of Schizosaccharomyces pombe rph16+, a gene homologous to the Saccharomyces cerevisiae RAD16 gene.

The RAD16 gene is involved in the nucleotide excision repair of UV damage in the transcriptional silenced mating type loci (Terleth et al., 1990 and Bang et al., 1992) and in non-transcribed stands of active genes in Saccharomyces cerevisiae (Verhage et al., 1994). Using touchdown-PCR with primers derived from various domains of the S. cerevisiae Rad 16 protein, a specific Schizosaccharomyces pombe probe was isolated. This probe was used to obtain the complete RAD16 homologous gene from a S. pombe chromosomal bank. DNA sequence analysis of the rph16+ gene revealed an open reading frame of 854 amino acids. Comparison of the amino acid sequences of the Rhp16 and Rad16 proteins showed a high level of conservation: 68% similarity. The Rhp16 protein sequence contains the two Zn-finger motifs and the putative helicase domains as found in the Rad16 protein. Like the RAD16, the rph16+ gene is UV-inducible (Bang et al., 1995). In analogy with the rad16 mutant, the rhp16 disruption mutant is viable and grows normally, indicating that the gene does not have an essential function. The rhp16 disruption mutant is not sensitive for UV but is sensitive for cisplatin. The rhp16+ gene cloned behind the GAI 1 promoter partially complements the UV sensitivity and the defect in the non-transcribed strand DNA repair of a S. cerevisiae rad16 mutant, indicating functional homology between the rhp16+ and RAD16 genes. The structural and functional homology between the two genes suggests that the RAD16 dependent subpathway of NER for the repair of non-transcribed DNA is evolutionary conserved.

Adenosine Triphosphatases↗
PubMed1997

Analysis of genes for human snRNP Sm-D1 protein and identification of the promoter sequence which shows segmental homology to the promoters of Sm-E and U1 snRNA genes.

The Sm core proteins of U1, U2, U4/U6 and U5 snRNPs include B(B1), B'(B2), N(B3), D1, D2, D3, E, F and G polypeptides. We have isolated genomic clones encoding the Sm-D1 protein using the Sm-D1 cDNA as probe. Southern blotting and DNA sequencing analysis of these clones revealed the presence of an Sm-D1 multigene family in the human genome. Three gene members have been identified. Two of the genes are without introns and contain mutations compared to the cDNA sequence. They appear to be processed pseudogenes. The third gene, termed SNRPD1, shares 100% identity to the cDNA sequence including both 5'- and 3'-untranslated regions (UTR); it contains three introns. Analysis of the 5'-flanking region of the SNRPD1 gene revealed promoter activity, suggesting this is the functional gene that encodes the Sm-D1 protein. The promoter activity was localized in a 0.38 kb PstI fragment using CAT reporter gene fusion assays. Addition of an SV40 enhancer element did not enhance the transcription directed by that fragment. Sequence comparison of the 0.38 kb promoter sequence with the promoters of the Sm-E gene and U1 snRNA genes revealed several homologous motifs, suggesting that genes encoding the snRNP components may be coordinately regulated.

Amino Acid Sequence↗
PubMed2001

Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma.

Epithelial ovarian carcinoma is often diagnosed at an advanced stage of disease and is the leading cause of death from gynaecological neoplasia. The genetic changes that occur during the development of this carcinoma are poorly understood. It has been proposed that IGFIIR, TGFbeta1 and TGFbetaRII act as a functional unit in the TGFbeta growth inhibitory pathway, and that somatic loss-of-function mutations in any one of these genes could lead to disruption of the pathway and subsequent loss of cell cycle control. We have examined these 3 genes in 25 epithelial ovarian carcinomas using single-stranded conformational polymorphism analysis and DNA sequence analysis. A total of 3 somatic missense mutations were found in the TGFbetaRII gene, but none in IGFRII or TGFbeta1. An association was found between TGFbetaRII mutations and histology, with 2 out of 3 clear cell carcinomas having TGFbetaRII mutations. This data supports other evidence from mutational analysis of the PTEN and beta-catenin genes that there are distinct developmental pathways responsible for the progression of different epithelial ovarian cancer histologic subtypes.

Carcinoma↗
PubMed1997

The preferential mode analysis of DNA sequence.

After reviewing approaches to the nucleotide correlation of DNA sequences the preferential mode analysis method is emphasized and discussed in detail. The preferred modes and poor modes in coding regions, as well as in introns, 5'-caps and 3'-tails are found through the statistical analysis of sequence data of all kinds of species in GenBank. The relation between the preferential mode analysis and informational parameter method is deduced. It is discovered that in higher species the coding sequences preferentially use the strong-weak bond (strong bond=C,G; weak bond=A, T) language and many noncoding regions (introns, 5'-caps, 3'-tails) use purine-pyrimidine language. The application of different languages in coding and noncoding sequences is a result of evolution, and it may be related to the functional differences in these two regions. Furthermore, we find that many preferential triplets in coding sequences can be expressed in a form of (* W S) (W=A,T; S=C,G), which may be explained by its relation to t-RNA abundance. The systematic change of some mode contents with evolution has also been found.

Animals↗
PubMed1996

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state.

Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome 1. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings.

Aerobiosis↗
PubMed2002

Expression pattern of alternatively spliced PECAM-1 isoforms in hematopoietic cells and platelets.

PECAM-1 (CD31) is a cell adhesion molecule that is highly expressed in the endothelium. Hematopoietic cells including platelets, monocytes, neutrophils, and some T cells also express moderate levels of PECAM-1. PECAM-1 undergoes alternative splicing generating a number of isoforms in the endothelium. However, the expression of PECAM-1 isoforms in hematopoietic cells and platelets has not been determined. Here, we examined the expression pattern of PECAM-1 isoforms in human and rodent hematopoietic cells and platelets by RT-PCR and DNA sequencing analysis. Our results showed that multiple PECAM-1 isoforms are expressed in a cell-type and species-specific pattern. We identified seven human PECAM-1 isoforms, six murine PECAM-1 isoforms, and four rat PECAM-1 isoforms. The full-length PECAM-1 was the predominant isoform detected in human cells. The PECAM-1 isoforms that lack exon 14 and 15 (delta14&15) or delta12,14&15 were the predominant isoform in rodent cells. In addition, we identified a novel PECAM-1 isoform, delta13&14, in human hematopoietic cells. Thus, hematopoietic cells express multiple isoforms of PECAM-1 in a pattern similar to that observed in the endothelium of the same species. The regulated expression of these isoforms may be important during hematopoiesis and transendothelial migration.

Alternative Splicing↗
PubMed1998

Cloning of a novel member of the low-density lipoprotein receptor family.

A gene encoding a novel transmembrane protein was identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation.

Amino Acid Sequence↗
PubMed1996

Selective expansion of specific T cell receptors in the inflamed colon of Crohn's disease.

To identify disease-specific T cell changes that occur in Crohn's disease (CD), the T cell receptor BV repertoires of lamina propria lymphocytes (LPL) isolated from both the inflamed and "disease-inactive" colons of seven CD patients were compared by the quantitative PCR and DNA sequence analysis. It was observed that the BV repertoires of LPL isolated from the disease-active and disease-inactive parts of the colon from the same individual were very different. Furthermore, nearly all of the differences occurred in CD4+ LPL, with very few differences in the CD8+ population of LPL. Although the pattern of BV segments that was increased in disease-active tissue relative to disease-inactive tissue was different for all seven CD patients, there were several BV segments that increased uniformly in the disease-active tissue of all seven individuals. CDR3 length analysis and DNA sequencing of these BV segments revealed that in six of the seven CD patients there was a striking degree of oligoclonality that was absent from disease-inactive tissue of the same individual. These observations suggest that at least some of the inflammation in CD is the result of responses by CD4+ T cells to specific antigens. The isolation of such inflammation-specific CD4+ T cells may make it possible to identify the antigens that are responsible for the inflammatory process in CD and provide a better understanding of its pathogenesis.

Adolescent↗
PubMed2002

Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation.

Carotenoids synthesized in plants but not animals are essential for human nutrition. Therefore, ongoing efforts to metabolically engineer plants for improved carotenoid content benefit from the identification of genes that affect carotenoid accumulation, possibly highlighting potential challenges when pyramiding traits represented by multiple biosynthetic pathways. We employed a heterologous bacterial system to screen for maize cDNAs encoding products that alter carotenoid accumulation either positively or negatively. Genes encoding carotenoid biosynthetic enzymes from the bacterium Erwinia uredovora were introduced into Escherichia coli cells that were subsequently transfected with a maize endosperm cDNA expression library; and these doubly transformed cells were then screened for altered carotenoid accumulation. DNA sequencing and characterization of one cDNA class conferring increased carotenoid content led to the identification of maize cDNAs encoding isopentenyl diphosphate isomerase. A cDNA that caused a reduced carotenoid content in E. coli was also identified. Based on DNA sequence analysis, DNA hybridization, and further functional testing, this latter cDNA was found to encode the small subunit of ADP-glucose pyrophosphorylase, a rate-controlling enzyme in starch biosynthesis that has been of interest for enhancing plant starch content.

Amino Acid Sequence↗
PubMed1998

Structure of the human D1F15S1A locus: a chromosome 1 locus with 97% identity to the chromosome 3 gene coding for hepatocyte growth factor-like protein.

The human chromosome 3 locus coding for hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL/MSP) is homologous to two sets of amplified loci on human chromosome 1 at 1p36. One copy of one of the amplified loci (D1F15S1A) has been further characterized by restriction enzyme and DNA sequence analysis. A total of 8331 bp of continuous sequence was determined for this locus. The first 6878 bp of sequence is 96.1% identical to the HGFL/MSP gene, while there is no homology between the two genes following nucleotide 6878. Based on the presence of a 5 bp deletion in putative exon 2 and several downstream stop codons it is very likely that this gene is a pseudogene. Screening of a human liver cDNA library with a chromosome 1-specific probe indicates that at least several other members of the chromosome 1 loci are transcribed.

Chromosomes, Human, Pair 1↗
PubMed1995

Sequence analysis of leukotoxin secretion determinants from a Pasteurella haemolytica-like organism.

The pHLBD genes encoding the secretion functions for the 105 kDa RTX leukotoxin of Pasteurella haemolytica-like (PHL) organism has been cloned and sequenced. Like analogous genes from other RTX determinants, the pHLBD genes lie immediately downstream from the leukotoxin structural gene, pHLA. Although isolated from a diverse group of gram-negative organisms, the pHLBD genes and the characterized RTX BD genes from other organisms exhibit a high degree of homology at both the DNA and predicted amino acid sequence levels. We have previously reported the cloning of the leukotoxin gene (pHLCA) (Chang et al., Infect. Immun. 61:2089-2095), which encodes a 105-kda polypeptide with cytotoxic activity. DNA sequence analysis of the pHLBD genes shows 83.93% and 86.05% homologous to that of P. haemolytica IktBD genes, respectively.

ATP-Binding Cassette Transporters↗