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PubMed1979

Variability of bacterial gene-directed enzyme production in human genetically deficient cells.

Human beta-galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GMI-gangliosidosis type I) were treated with phage lambda plac DNA, coding for Escherichia coli beta-galactosidase (beta-D-galactoside galactohydrolase, EC.3.2.1.23). New beta-galactosidase activity detected in cell extracts of phage DNA-treated GMI-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant beta-galactoside activity in lambda plac DNA-treated cells resembled that of mutant E. coli beta-galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3'-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions. More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q beta-replicase, f 1-coat protein, or UDPG-4-epimerase.

Cells, Cultured↗
PubMed2002

High expression of vascular endothelial growth factor predicts early recurrence and poor prognosis after curative resection for ductal adenocarcinoma of the pancreas.

INTRODUCTION AND AIMS: Only curative resection for pancreatic adenocarcinoma is related to a favorable prognosis, but the overall survival after surgery still remains poor, and early recurrence is frequently observed. Because recurrence is the limiting factor and the main cause of death after curative resection, the identification of markers that predict early postoperative recurrence is of paramount importance. Angiogenesis is essential for tumor growth and metastases; therefore, we set out to clarify whether vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) correlate with early recurrence and poor prognosis after curative resection. A second goal was to characterize the VEGF-producing cells and the subcellular distribution. METHODOLOGY: Seventy patients with ductal adenocarcinoma of the pancreas were studied after curative resection with a follow-up of at least 2 years. The MVD quantification was performed immunohistochemically with use of a monoclonal antibody to CD34. The VEGF expression was studied with use of polyclonal antibody. To detect the intracellular localization of specific VEGF mRNA sequences, nonisotopic in situ hybridization was performed. The correlations among VEGF expression and MVD, clinicopathologic parameters, and clinical outcome were then statistically analyzed. RESULTS: The VEGF immunoreactivity was 88.6%, and positive mRNA signals were obtained in the cytoplasm of carcinoma and endothelial cells in 81.4%. Furthermore, we observed tumor-associated macrophages close to infiltrating carcinoma cells. All endothelial cells showed positive immunoreactivity to the anti-CD34 antibody, and a median distribution of 85 vessels/x200 field was observed. A significant correlation (p < 0.05) was found between the MVD and the International Union Against Cancer (UICC) stage. Statistical analysis showed a significant correlation between VEGF expression and the height of MVD (p < 0.05). Kaplan-Meier analyses revealed that VEGF expression and MVD had a statistically significant correlation with survival after curative resection (p < 0.05). Furthermore, multivariate analysis indicated that VEGF expression is an independent prognostic marker for cancer recurrence within 8 months after curative surgery (p = 0.003). CONCLUSION: In pancreatic adenocarcinoma, the VEGF expression and the height of MVD are closely correlated, and both-rather than UICC stage and TNM classification (tumor size and nodal involvement)-are markers of prognostic relevance after curative resection. Furthermore, VEGF is a predictor of early recurrence after curative resection. The current study indicates that VEGF may promote the distribution of metastases, leading to early cancer recurrence and poor outcome.

Antigens, CD34↗
PubMed2001

Mapping of genes and transcribed sequences in a gene rich 400-kb region on human chromosome 11p15.1-->p14.

We have identified a number of transcribed sequences within a 400-kb interval on chromosome 11p15.1--> p14. Six genes and 13 novel transcripts including ESTs, cDNAs and exons have been identified and assigned to this region. Comparison of mRNA sequence with genomic sequence has enabled us to determine the exon/intron structure of four of the genes (NUCB2, PIK3C2A, RPS13 and OR7E14P).

Base Sequence↗
PubMed1990

The structurally similar neuropeptides adipokinetic hormone I and II are derived from similar, very small mRNAs.

The grasshopper neuropeptides adipokinetic hormone (AKH) I and II were among the first of an extensive family of structurally similar arthropod hormones and neuroregulators to be isolated and sequenced. This paper reports the cloning of cDNAs derived from the unusually small mRNAs (550 bases) which code for the precursors of AKH I and II from Schistocerca nitans. Sequence analysis of the cDNAs indicates that AKH I and II are derived from small precursor proteins (63 and 61 amino acids) which are 55% identical in amino acid sequence. Each contains a 22-amino acid hydrophobic leader sequence followed by the AKH I or II sequence and an additional 28-amino acid carboxyl-terminal peptide of unknown function. Significant homology at the nucleic acid level (64% identity) is confined to the coding region of the mRNA sequences. Preliminary DNA blot analyses suggest that a single gene codes for each, and that the genes for AKH I and II may be linked. Genomic blots from various tissues fail to suggest that the high level of expression of AKH in the corpora cardiaca is due to tissue specific gene amplification.

Amino Acid Sequence↗
PubMed1983

The complete nucleotide sequence of the major adult beta globin gene of Xenopus laevis.

We present the complete nucleotide sequence of the major beta globin gene of Xenopus laevis with 127 nucleotides of sequence upstream of the gene and 212 nucleotides downstream. The start point of transcription was determined by S1 mapping and primer extension and the site of polyadenylation by comparison with the mRNA sequence determined from a cDNA clone. Comparison of the X. laevis sequence with other genes transcribed by RNA polymerase II reveals conserved features in the 5' flanking, 5' noncoding, intron/exon boundary, and 3' noncoding regions.

Amino Acid Sequence↗
PubMed1993

Mitochondrial cytochrome C oxidase II messenger ribonucleic acid is expressed in pachytene spermatocytes at high levels and in a stage-dependent manner during spermatogenesis in the rat.

Screening of a rat seminiferous tubule library (stages VI-VIII) resulted in isolation of several clones to the same abundant mRNA. Sequencing revealed that these encoded all or part of the second subunit of cytochrome C oxidase (COX II), the terminal enzyme in the electron transport chain located in mitochondria. The pattern of expression of the COX II mRNA in the testis was examined by Northern blot and in situ hybridisation combined with specific depletion of pachytene spermatocytes or Leydig cells by treatment with methoxyacetic acid (MAA) or ethane dimethane sulphonate (EDS), respectively. COX II mRNA was found to be strongly expressed in pachytene spermatocytes in adult and prepubertal rats, with the highest levels of expression at stages VII-VIII of the spermatogenic cycle. Although this is the androgen-dependent stage, Northern analysis of total testicular RNA showed that expression of COX II mRNA was not altered detectably by EDS-induced androgen withdrawal nor was expression altered detectably by 24-h treatment with high doses of FSH. Treatment of rats with MAA, which selectively depletes seminiferous tubules at most stages of pachytene spermatocytes, resulted in a marked reduction in COX II mRNA. In the absence of pachytene spermatocytes, COX II mRNA was visualized in preleptotene spermatocytes and in Sertoli cell cytoplasm. The expression of COX II in Sertoli cells was confirmed by examination in situ of a testis devoid of most germ cells and by Northern analysis of RNA from an adult Sertoli cell-enriched preparation. However, using either approach the amount of mRNA in Sertoli cells was considerably lower than that in pachytene spermatocytes. Cytochrome C oxidase has thirteen subunits, three of which (I-III) are encoded on the mitochondrial genome. The physiological significance of the high levels of expression of COX II mRNA in pachytene spermatocytes and its stage-dependent changes is unknown but they presumably reflect requirements for alterations in energy demand as these cells enter the final stages of meiosis. The stage-dependent differences in expression of COX II mRNA in pachytene spermatocytes may also explain differences in the susceptibility of these cells to depletion by MAA.

Animals↗
PubMed1988

Multiple protein 4.1 isoforms produced by alternative splicing in human erythroid cells.

Protein 4.1 is a multifunctional structural protein located in the erythrocyte membrane skeleton and in many nonerythroid cells. Molecular characterization of cloned protein 4.1 sequences from human reticulocytes has revealed the existence of multiple transcripts of the protein 4.1 gene that may encode a family of closely related protein isoforms. Several independently isolated cDNAs were sequenced and demonstrated to encode four different protein 4.1 species having identical primary sequences, except for the presence or absence of discrete peptides in the 8-kDa spectrin/actin binding domain (21 amino acids) and near the carboxyl terminus (43 and 34 amino acids). The same four protein 4.1 isoforms were detected when reticulocyte protein 4.1 mRNA sequences were reverse transcribed into cDNA and enzymatically amplified in vitro by using protein 4.1-specific oligonucleotide primers and the polymerase chain reaction. The finding of multiple protein 4.1 isoforms raises the possibility that the many binding functions ascribed to protein 4.1 may reside in distinct structural isoforms. Since only a single protein 4.1 gene appears to be expressed in erythrocytes, it is likely that these isoforms are produced by alternative mRNA splicing from a common protein 4.1 pre-mRNA. Multiple RNA splicing pathways are thus operative in the protein 4.1 gene even within a single cell lineage, human erythroid cells.

Amino Acid Sequence↗
PubMed1991

Molecular approaches for analyzing differential gene expression: differential cDNA library construction and screening.

Complementary deoxyribonucleic acid (cDNA) libraries can be used as a means to isolate and identify cell-specific messenger ribonucleic acid (mRNA) sequences. The basic elements of cDNA library construction and screening are reviewed in the context of analyzing differentially expressed mRNAs. A brief overview of the recombinant DNA systems applied to cDNA library construction and the principles of screening cDNA libraries by plaque hybridization are provided. Methods for comparing mRNA populations by differential screening and by competition hybridization are discussed, and methods for constructing subtracted cDNA libraries, enriched in differentially expressed sequences, are presented. Also reviewed are the analysis of differentially expressed cDNAs by Southern and Northern hybridization, RNase protection, polymerase chain reaction, and sequencing.

Animals↗
PubMed1991

Maple syrup urine disease. Complete defect of the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase complex due to a deletion of an 11-bp repeat sequence which encodes a mitochondrial targeting leader peptide in a family with the disease.

Branched chain alpha-ketoacid dehydrogenase (BCKDH) deficiency results in maple syrup urine disease (MSUD). We examined the molecular basis of familial cases of MSUD by analyzing the activity, subunit structure, mRNA sequence, and genome structure of the affected enzyme. The BCKDH activity in the proband with MSUD was approximately 6% of the normal control level. Immunoblot analysis revealed that the E1 beta subunit of BCKDH was absent and that the E1 alpha subunit of BCKDH was markedly reduced. We amplified the cDNAs of the E1 alpha subunit and the E1 beta subunit of the BCKDH complex obtained from cells of the patient, using the polymerase chain reaction method, then sequenced the amplified cDNAs. The deduced amino acid sequence for the E1 alpha subunit of the patient's cell was normal. An 11-bp deletion was identified in the region that encoded the mitochondrial targeting leader peptide in the E1 beta cDNA. This 11-bp sequence is found in the first exon of the BCKDH-E1 beta gene, as a direct tandem repeat. Amplification of genomic DNA revealed that the consanguineous parents were heterozygous for this mutant allele, and sister and brother of the patient with the disease were homozygous for this mutant allele. This 11-bp deletion mutation caused a change in the reading frame and the mature E1 beta protein was defective. These observations show the biological importance of the E1 beta subunit of BCKDH to maintain normal function of the enzyme activity. The absence of the E1 beta subunit results in instability of the E1 alpha subunit.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1988

Identification of a human glial fibrillary acidic protein cDNA: a tool for the molecular analysis of reactive gliosis in the mammalian central nervous system.

Two clones encoding human glial fibrillary acidic protein (GFAP) were isolated from a human astrocytoma cDNA library. The clones pHGFAP1 and pHGFAP2 were selected by the combined use of differential colony hybridization and hybridization-selection technique with polyclonal anti GFAP antiserum. The longer one, pHGFAP1, encompasses 3.0 kb and includes the 1.8 kb long 3' untranslated region specific to the human mRNA. Sequence data disclosed an extensive homology within the coding region of human and mouse GFAP cDNAs even in the end domains. Blot hybridization analysis of RNAs from human, rat and mouse brain revealed a single GFAP mRNA species of 3.1, 2.8 and 2.7 kb respectively and Southern blot experiments indicated that this mRNA is most probably transcribed from a unique gene. In situ hybridization performed with biotinylated probes on cultured mouse brain cells suggests both the sorting and the transport of GFAP mRNA throughout the cytoplasm and processes of the astrocytes. As a model of reactive gliosis secondary to degenerative disorders, 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra in the rat was performed. GFAP mRNA increased 1.4 fold in the ipsilateral striatum on day 10 after the lesion. It then declined to the control level 4 months later contrasting with the lower and more sustained increase in preproenkephalin (PPE) mRNA. The interspecies cross-reactivity of the HGFAP probes make them useful as a tool for the molecular analysis of reactive gliosis in various experimental models.

Amino Acid Sequence↗
PubMed1987

The isolation and characterisation of cDNA and genomic clones for human lecithin: cholesterol acyltransferase.

The protein sequencing of tryptic peptides from purified human lecithin: cholesterol acyltransferase (LCAT) identified sufficient amino-acid sequence to construct a corresponding mixed oligonucleotide probe. This was used to screen an adult human cDNA liver library, from which incomplete cDNA clones were isolated. The DNA sequence of these clones allows the prediction of the entire amino-acid sequence of the mature LCAT enzyme. The mature protein consists of 416 amino acids and contains several marked stretches of hydrophobic residues and four potential glycosylation sites. The cDNA probe detects LCAT mRNA sequences approx. 1500 bases long in human liver, but not intestine, RNA. The cDNA probe was used to isolate LCAT genomic recombinants from a human genomic library. Southern blotting data, and restriction site mapping, suggest that there is a single human LCAT structural gene between 4.3 and 5.5 kb in size.

Amino Acid Sequence↗
PubMed2003

A double-screening method to identify reliable candidate non-synonymous SNPs from chicken EST data.

Discovery of non-synonymous single nucleotide polymorphisms (nsSNP), which cause amino acid substitutions, is important because they are more likely to alter protein function than synonymous SNPs (sSNP) or those SNPs that do not result in amino acid changes. By changing the coding sequences, nsSNP may play a role in heritable differences between individual organisms. In the chicken and many other vertebrates, the main obstacle for identifying nsSNP is that there is insufficient protein and mRNA sequence information for self-species referencing and thus, determination of the correct reading frame for expressed sequence tags (ESTs) is difficult. Therefore, in order to estimate the correct reading frame at nsSNP in chicken ESTs, a double-screening approach was designed using self- or cross-species protein referencing, in addition to the ESTScan coding region estimation programme. Starting with 23 427 chicken ESTs, 1210 potential SNPs were discovered using a phred/phrap/polyphred/consed pipeline process and among these, 108 candidate nsSNP were identified with the double screening method. A searchable SNP database (chicksnps) for the candidate chicken SNPs, including both nsSNPs and sSNPs is available at http://chicksnps.afs.udel.edu. The chicken SNP data described in this paper have been submitted to the data base SNP under National Center for Biotechnology Information assay ID ss4387050-ss4388259.

Animals↗
PubMed1979

Molecular cloning and nucleotide sequence of the human growth hormone structural gene.

An almost complete cDNA copy of human growth hormone has been cloned and sequenced. The nucleotide sequence confirms the known protein sequence and predicts the sequence of a precursor region of 26 amino acids. We have compared the nucleotide sequence to that for the homolgous proteins, rat growth hormone and human chorionic somatomammotropin (Seeburg et al. and Shine et al., Nature 270, 486 (1977)). There appears to be evolutionary conservation of mRNA sequence features not related to protein structure.

Amino Acid Sequence↗
PubMed2004

Predicting genes expressed via -1 and +1 frameshifts.

Computational identification of ribosomal frameshift sites in genomic sequences is difficult due to their diverse nature, yet it provides useful information for understanding the underlying mechanisms and discovering new genes. We have developed an algorithm that searches entire genomic or mRNA sequences for frameshifting sites, and implements the algorithm as a web-based program called FSFinder (Frameshift Signal Finder). The current version of FSFinder is capable of finding -1 frameshift sites on heptamer sequences X XXY YYZ, and +1 frameshift sites for two genes: protein chain release factor B (prfB) and ornithine decarboxylase antizyme (oaz). We tested FSFinder on approximately 190 genomic and partial DNA sequences from a number of organisms and found that it predicted frameshift sites efficiently and with greater sensitivity and specificity than existing approaches. It has improved sensitivity because it considers many known components of a frameshifting cassette and searches these components on both + and - strands, and its specificity is increased because it focuses on overlapping regions of open reading frames and prioritizes candidate frameshift sites. FSFinder is useful for discovering unknown genes that utilize alternative decoding, as well as for analyzing frameshift sites. It is freely accessible at http://wilab.inha.ac.kr/FSFinder/.

Algorithms↗
PubMed2005

Sequence-based discovery of the human and rodent peroxisomal proteome.

BACKGROUND: Peroxisomes are metabolic organelles present in virtually all eukaryotic cells. They contain enzymes involved in hydrogen peroxide-based respiration and lipid metabolism. At present, only a small number of peroxisomal enzymes that are associated with oxidative stress response and metabolic disorders have been characterised biochemically. Therefore, we devised a sequence-based, multistep knowledge discovery strategy to identify potential novel peroxisomal protein candidates in small rodent model organisms and human. METHODS: Screening of 130,629 putative translations of GenBank rodent and primate mRNA sequences was limited to the classical type-1 peroxisomal targeting signal [SA]-K-L. This motif is over-represented among peroxisomal proteins and has a high targeting efficiency. Subsequent steps of identifying co-occurring motifs, secondary structure properties, orthologues and variants, in combination with literature searching and visual inspection by domain experts, aimed at reduction of both false positive and negative validation targets. RESULTS: Our method yielded 117 known peroxisome-targeted proteins and 29 novel candidate proteins. Of special interest were the mouse C530046K17Rik and 1300019N10Rik protein sequences that contain domains associated with enzymatic functions. C530046K17Rik showed no similarity to any known sequence of the animal kingdom, but weak similarity to the possible Leishmania quinone oxidoreductase and a putative cyanobacterium nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidoreductase. 1300019N10Rik contains two protease-related domains, glutamyl endopeptidase I and trypsin-like serine and cysteine proteases, which may have unique specificities to achieve efficient breakdown of proteins in the peroxisomes. CONCLUSION: One mouse C57BL/6J strain-specific isocitrate dehydrogenase 1 isoform might be suitable to investigate potential phenotypes associated with the deficit of the intraperoxisomal reduced form of NADP (NADPH) and 2-oxoglutarate. Our biological knowledge discovery strategy enabled not only the identification of peroxisomal enzymes already described in the literature, but also the prediction of several novel proteins with possible roles in peroxisomal biochemistry and metabolism that are currently under experimental validation.

Amino Acid Sequence↗
PubMed2002

[Molecular cloning and analysis of bovine prolactin full-long genomic as well as cDNA sequences].

In this report, a full-length sequence of bovine prolactin (bPRL) genomic DNA with 9388 bp, which has been accepted by GenBank (Accession Number: AF426315), was firstly cloned by Long PCR procedures. This sequence consists of 5 exons, 4 introns, 854 bp of 5' upstream regulatory region and 69 bp of 3' UTR. Accession number of protein encoded by AF426315 sequence in GenBank is AAL28075 that is composed of 229 amino acid residues, in which signal sequence resides in 1-30 sites and mature polypeptide consists of 199 amino acid residues. The recombinant plasmid containing bPRL genomic DNA was then transfected into eukaryotic cells (COS-7), followed by RT-PCR procedure. An 804 bp of bPRL cDNA containing all the encoding region was obtained, indicating that the bPRL genomic DNA reported herein had its biological function at the transcriptional level. Results derived from information searching by Blast program revealed that there were various SNP sites in the sequences of bPRL mRNAs and ESTs collected in GenBank, which located mainly in downstream encoding region and 3' UTR. These SNP sites did not alter the related amino acids encoded. In addition, mRNA sequences encoding 5' signal sequence of bPRL gene was highly conserve.

Animals↗
PubMed1995

Cloning novel alfalfa cyclin sequences--a RACE-PCR approach.

Cyclins are a complex group of proteins involved in regulation of the eukaryotic cell division cycle via their interaction with cyclin dependent kinases (Cdks). Cyclin gene sequences have been cloned from a number of plant species, including alfalfa, but the diversity of these genes suggests that there are many plant cyclins which have yet to be characterized. A RACE-PCR strategy has been adopted for cloning cyclin gene sequences expressed during direct somatic embryogenesis in alfalfa. RT-PCR with nested degenerate primers was used to amplify the highly conserved "cyclin box" region of a novel A-like cyclin mRNA sequence expressed after induction of somatic embryogenesis. The sequence of this PCR product was used to design primers for 5'- and 3'-RACE protocols. 5'-RACE using a modified SLIC (single strand ligation to single stranded cDNA) procedure revealed considerable sequence heterogeneity in the N-terminal region of the coding sequence with several closely related sequences apparent. Conventional 3'-RACE generated a single cyclin sequence. The complete coding sequence of one member of this A-like cyclin subgroup has been obtained by this RACE strategy and confirmed by PCR amplification and sequencing of alfalfa genomic DNA.

Amino Acid Sequence↗
PubMed1991

Tumor necrosis factor-alpha mRNA accumulation in human myelomonocytic cell lines. Role of transcriptional regulation by DNA sequence motifs and mRNA stabilization.

The cytokine TNF mediates many of the pathologic signs of cachexia, inflammation, and sepsis. The current work describes the regulation of TNF in human myelomonocytic cell lines after PMA stimulation. The cell lines exhibit a low level of constitutive TNF mRNA expression. Within 2 to 4 h of PMA exposure, steady state levels of TNF mRNA are markedly elevated in all myelomonocytic cell lines studied. This rise is due to increased mRNA stability, which increased by almost twofold, and to an overall increase in transcription, which rises by more than sixfold. At the level of the genomic TNF gene, a DNase I hypersensitive site is detected within the TNF promoter between -200 to -100 bp relative to the transcription initiation site. Although absent in nonexpressing erythroleukemia cell lines, the DNase I site is present in uninduced myelomonocytic cell lines and is not changed after PMA induction. The PMA induction of c-fos mRNA correlated well with TNF gene induction; expression of genes encoding other proteins in the AP-1 complex (junB and junD) were also induced by PMA. The nuclear extracts from resting and induced ML-1 cells contain proteins binding specifically to the AP-1, AP-2, and NF kappa B sequence located within the TNF promoter. PMA induction increases the level of a number of specific binding complexes relative to the resting cells. The regulatory mechanisms of the human and murine TNF genes are discussed.

Base Sequence↗