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PubMed2002

Novel alleles at the JK blood group locus explain the absence of the erythrocyte urea transporter in European families.

The Kidd (JK) blood group system is of importance in transfusion medicine. The Jk(null) phenotype is associated with absence of the urea transporter in erythrocytes and moderately reduced ability to concentrate urine. We and others recently reported different molecular alterations in the silenced Jkb-like alleles of Polynesians and Finns, populations with higher Jk(null) frequencies. Here we report novel molecular bases of this phenotype in Caucasians. Blood samples from a Swiss and an English family were investigated by serological methods, urea haemolysis test and JK genotyping. Genomic DNA and JK mRNA were sequenced. Genotyping showed homozygosity for Jka-like alleles. The Swiss Jk(null) alleles deviated from wild-type Jka sequence by a nonsense mutation in exon 7 causing an immediate stop codon (Tyr194stop). The English Jk(null) alleles revealed a genomic 1.6 kilobase pair deletion including exons 4 and 5, the former of which includes the translation start codon. Multiple mRNA splicing variants were detected in reticulocytes but exons 3-5 were absent in all transcripts analysed. Screening for these alleles was negative in random donors. Two novel molecular alterations at the JK locus were defined and a multiplex polymerase chain reaction method for detection of the five known silent Jk alleles was developed to complement JK genotyping in clinical transfusion medicine.

Alleles↗

PubMed1996

Characterization of the 1B promoter of fibroblast growth factor 1 and its expression in the adult and developing mouse brain.

The present study elucidates the molecular structure of a murine fibroblast growth factor 1 (FGF-1) promoter and describes its distribution in the adult and developing mouse brain. A cDNA clone coding for FGF-1 was isolated from a mouse brain cDNA library. Nucleotide sequence analysis revealed that the clone contained, in addition to the protein coding region, an untranslated exon (FGF-1B) 34 base pairs upstream of the translation start codon ATG. The mouse cDNA clone corresponded to the sole FGF-1 transcript in the brain. An RNase protection assay was used to map the transcription start site of the 1B promoter. The sequences upstream from the major transcription initiation site lacked consensus TATA or CAAT boxes. In situ hybridization with cRNA probes specific for the 1B transcript showed the message to be restricted largely to sensory and motor nuclei in the brainstem, and to the ventral spinal cord and cerebellum. Although occasional brainstem nuclei were labeled at low levels by embryonic day 18, the majority of nuclei became detectable autoradiographically during postnatal weeks 1 and 2, and adult levels of grain density were reached during the 3rd and 4th postnatal weeks. FGF-1B mRNA was expressed in phylogenetically older brain regions, which are involved primarily in processing information from exteroceptive sensory mechanoreceptors and in motor control. The relatively late developmental expression suggests a role for FGF-1 in neuronal maturation, rather than in neurogenesis.

Aging↗

PubMed2004

Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes.

A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes--aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis--were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium.

Adaptation, Physiological↗

PubMed1994

The large surface protein of duck hepatitis B virus is phosphorylated in the pre-S domain.

The two major envelope proteins (large [L] and small [S]) of duck hepatitis B virus are encoded by the pre-S/S open reading frame. The L protein is initiated from the AUG at position 801 in the pre-S region of the pre-S/S coding sequence, yielding an N-terminal consensus sequence for myristylation. Western immunoblots of the L protein often reveal a doublet at 36 and 35 kDa, with the latter attributed to the use of one of the three internal initiation codons. However, metabolic labelling with [3H]myristic acid results in labelling of both P35 and P36, indicating that both species must be initiated from the same start codon. Using metabolic labelling with 32P and digestion with residue-specific phosphatases, we demonstrate that L protein heterogeneity is due to phosphorylation of threonine and/or serine residues within the pre-S domain. We propose that at least one possible phosphorylation site is located at a novel (S/T)PPL motif which is conserved near the carboxyl end of the pre-S1 domain in all hepadnavirus sequences. Two to three additional (S/T)P motifs are also present in the carboxyl half of the pre-S1 (but not pre-S2 or S) domain of all hepadnaviruses. L protein in serum-derived particles is resistant to phosphatase digestion in the absence of detergents, reflecting an internal disposition of the phosphorylated pre-S domain and suggesting a role for dephosphorylation in the topological shift within L during morphogenesis (P. Ostapchuk, P. Hearing, and D. Ganem, EMBO J. 13:1048-1057, 1994). Furthermore, we observe that the relative amount of the phosphorylated form of L increases with time in the viral growth cycle. These findings imply that phosphorylation-dephosphorylation of the L protein is an important, regulated mechanism necessary for correct virion morphogenesis.

Animals↗

PubMedDate not supplied

[Analysis of the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon].

The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli. It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon. The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed. The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes. For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001). These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E. coli.

Base Sequence↗

PubMed1995

Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda.

Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification.

Amino Acid Sequence↗

PubMed2001

Translational properties of mHNA, a messenger RNA containing anhydrohexitol nucleotides.

Short messenger RNAs (mRNAs) with hexitol residues in two codons were constructed and their properties were studied in an Escherichia coli in vitro translation system. The replacement of the natural ribonucleotides of mRNA in the AUG start codon and the UUC second codon by hexitol nucleotides did not influence the main steps of translation, as indicated by the same level of binding of mRNA with or without hexitol residues under P-site conditions, and the same yield of tRNA binding to the P- and A-sites. Moreover, both peptide formation and translocation took place on mRNAs with hexitol residues. The presence of an A-type messenger hexitol nucleic acid (mHNA)-transfer RNA (tRNA) duplex is important for efficient translation and the 2'-OH function in mRNA is not necessary for binding and movement through the ribosome. Groove shape recognition of the codon-anticodon complex, more than hydrogen-bond interactions of ribose residues in mRNA, is an important factor for correct translation.

Base Sequence↗

PubMed1986

Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase.

Amino Acid Sequence↗

PubMed1990

Functional analysis of the 5' regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase gene.

A functional analysis of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase (6-HDNO) gene promoter (-35 region TTGACA and -10 region TATCAAT) and the UUG translation start codon was performed using site-directed mutagenesis. Deletion of the C residue from the -10 promoter region or mutations introduced upstream of the -10 region resulted in an increased 6-HDNO expression in Escherichia coli cells in vivo and in both E. coli and A. oxidans coupled transcription-translation systems in vitro. From the identical behaviour of 6-HDNO promoter mutants in the heterologous and homologous systems, it is concluded that A. oxidans harbours an RNA polymerase functionally homologous to the E. coli sigma 70 and Bacillus subtilis sigma 43 polymerases. Replacement of the TTG codon (UUG translation initiation codon) with ATG led to a 3.7-fold increase in 6-HDNO expression in E. coli. This effect was less pronounced at higher promoter strengths, from 3.7 in the case of the 6-HDNO wild-type promoter, to 2.5 in the case of the consensus -10 region and to 1.7 in the case of the tac promoter. A double point mutation introduced close to the ribosome binding site resulted in almost the same increase in 6-HDNO expression (3.1-fold) as the TTG-to-ATG exchange. The failure of cAMP to stimulate 6-HDNO expression in the A. oxidans system indicated that expression of this gene in stationary phase cells is not regulated by cAMP-catabolite repressore protein-mediated mechanism of catabolite repression.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthrobacter↗

PubMed1996

Antisense oligodeoxynucleotides against the BZLF1 transcript inhibit induction of productive Epstein-Barr virus replication.

Expression of the Epstein-Barr virus (EBV) BZLF1 gene product, ZEBRA, in latently infected cells is sufficient to induce the viral lytic cycle. The use of oligodeoxynucleotides complementary to the BZLF1 transcript was studied to inhibit this induction of productive viral replication. For this purpose, we employed oligodeoxynucleotides complementary to the translation initiation codons and their flanking sequences. Incubation of Akata cells with the 25-mer phosphodiester (PO)- or phosphorothioate (PS)-antisense oligodeoxynucleotides for 3 h before stimulation with anti-immunoglobulin G antibodies (anti-IgG) partially inhibited the anti-IgG-mediated induction of ZEBRA synthesis. Both the PO- and PS-antisense oligodeoxynucleotide treatments also suppressed the productive EBV replication (as measured by linear DNA production) in a dose-dependent manner, with much greater efficiency than did PO and PS-oligodeoxynucleotides with sense, reverse or random sequences of the same length. Another 20-mer antisense oligodeoxynucleotide complementary to sequences downstream of the translation initiation codons showed a similar inhibitory effect on EBV replication. However, the inhibition was considerably lower when the cells were treated with oligodeoxynucleotides complementary to sequences upstream of the start codons. These results indicate that BZLF1 antisense oligodeoxynucleotides inhibit the viral activation in a sequence-specific fashion. In the virus-producer cell line P3HR-1, the same PS-antisense oligodeoxynucleotides also partially suppressed the spontaneous viral replication after 6-10 days, substantially more than the PS-random oligodeoxynucleotides. Inhibition of BZLF1 appears to be sufficient to suppress the induction of EBV replication.

Antigens, Viral↗

PubMed2006

Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli.

The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions -152 and -195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.

Antigens, Bacterial↗

PubMed1996

The three dominant female-sterile mutations of the Drosophila ovo gene are point mutations that create new translation-initiator AUG codons.

The Drosophila ovo gene, which encodes a putative transcription factor (Ovo) with TFIIIA-like zinc fingers, is required for female germline survival and proper oogenesis. Three dominant female-sterile ovoD mutations cause ovarian abnormalities that define an allelic series, with ovoD1 displaying the stronger phenotype and ovoD3 the weaker. We report here that all three ovoD mutations are point mutations that create new in-frame methionine codons in the 5' part of ovo. There are two types of overlapping ovo transcription units, ovo alpha and ovo beta. By using various ovo-lacZ reporter genes, we determined that the long Ovo isoforms starting at methionine M1, present in transcripts ovo alpha, are expressed at low levels only in mature oocytes. Short Ovo isoforms are translated from methionine M373, the first in-frame start codon present in transcript ovo beta, and correspond to the activity defined by recessive loss of function ovo mutations. The new AUGs created in ovoD mutations all are located upstream of the M373 initiation site. Our results support the hypothesis that they can substitute for M373 as translation starts and initiate the synthesis of Ovo proteins that have extra amino acids at their N termini. We propose that premature expression of long Ovo protein isoforms occurs in ovoD mutants and interferes with wild-type Ovo function in controlling female germline differentiation.

Amino Acid Sequence↗

PubMed1999

Genomic organization and expression properties of the MtSucS1 gene, which encodes a nodule-enhanced sucrose synthase in the model legume Medicago truncatula.

We have isolated and sequenced a sucrose synthase (SucS) cDNA from the model legume Medicago truncatula. This cDNA (MtSucS1) contains an ORF of 2418 bp, coding for a protein of 805 amino acids with a molecular mass of 92.29 kDa. The deduced amino acid sequence shows significant homology to other plant sucrose synthases, in particular to the nodule-enhanced sucrose synthases from pea and broad bean. Northern analysis revealed that the corresponding gene shows a ten-fold higher expression level in root nodules than in uninfected root, stem and leaf tissues. SucS protein was detected in root nodules from a variety of legumes, including M. truncatula. Whereas only one SucS isoform was detectable in root nodules, an additional sucrose synthase of slightly larger molecular weight was present in uninfected root, stem and flower tissues of M. truncatula. From our expression and sequence data we infer that the MtSucS1 gene encodes a nodule-enhanced sucrose synthase in M. truncatula. Southern hybridization data indicate that MtSucS1 is a single-copy gene. An analysis of a genomic MtSucS1 sequence revealed that the gene consists of 14 exons with the start codon being located on exon II. As is common for SucS genes, the MtSucS1 gene contains a large intron of 747 bp in the 5' untranslated region. The transcriptional start of MtSucS1 was mapped and putative regulatory elements in the MtSucS1 promoter were identified.

Amino Acid Sequence↗

PubMed1992

Structure and chromosomal localization of the gene encoding barley seed peroxidase BP 2A.

A clone, lambda Prx6.1, coding for a barley seed peroxidase (BP; EC 1.11.1.7), was isolated from a genomic library using a cDNA coding for the barley seed peroxidase, BP 1, as a probe. The nucleotide sequence coded for a BP showing 73% amino acid (aa) sequence identity with BP 1 and less than 50% similarity with other sequenced plant peroxidases. The aa composition is 92% identical to that determined for BP 2 purified from mature barley grains, and therefore the gene product is named BP 2A. The alignment suggests that the coding region is interrupted by a 76-bp intron having the consensuses GT and AG, at the 5' and 3' ends, respectively. Alignment with BP 1 suggests that BP 2A has a leader peptide of 36 aa and the mature protein is 319 aa. Alanine and leucine account for 50% of the residues of the leader peptide. Of the codons used 90% have a C or G in the third position. The promoter shows a putative abscisic acid-response element, 5'-GTACGTGTC, 115 bp upstream from the start codon. The BP 2A-encoding gene was RFLP-mapped on barley chromosome 3, and we suggest for this peroxidase locus the name Prx6.

Amino Acid Sequence↗

PubMed1993

Codon preference in corynebacteria.

The codon usage (CU) of 34 genes from the closely related species, Brevibacterium lactofermentum and Corynebacterium glutamicum (BLCG), was analysed and compared with that of 23 genes from other Brevibacterium and Corynebacterium species. The G+C content of the BLCG genes ranged from 50 to 62%. A wider range was found in other corynebacterial genes (25-71%). The G+C contents of non-coding regions in glutamic acid bacteria are lower than those of the coding regions and both values are lower than the G+C content of ribosomal RNA (rRNA) sequences, suggesting an unusual biased mutation pressure. The CU and synonymous codon usage (SCU) analysis showed several common characteristics among the sequenced corynebacterial genes, consistent with the close relatedness of B. lactofermentum and C. glutamicum. A subset of 25 preferred codons were deduced from the presumably highly expressed genes and they encode most of the amino acid (aa) residues of the BLCG group. An analysis of the effective number of codons (Nc) was carried out in order to check the GC3s (G+C content at the silent third position of sense codons) dependence of the CU in corynebacteria. Nc values showed differences between the BLCG group and other corynebacterial sequences. A comparison of the most used codons for each aa showed a stronger similarity to Streptomyces than to Escherichia coli. The CU/SCU tables of corynebacteria are useful for identification of protein-coding regions, including start codons when they are uncertain, and for designing oligodeoxyribonucleotide probes from an aa sequence.

Base Sequence↗

PubMed1993

HIV1 integrase expressed in Escherichia coli from a synthetic gene.

Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two primers (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.

Amino Acid Sequence↗

PubMed1992

Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts.

Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the beta-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.

Base Sequence↗

PubMed1986

Isolation and characterization of the actin gene from Tetrahymena thermophila.

The macronucleus of Tetrahymena thermophila contains a single actin gene. We have isolated this gene from a partial plasmid library by using the yeast actin gene as a probe. The nucleotide sequence of the gene has been determined and the amino acid sequence of the potential protein deduced. The encoded protein is 375 amino acids long, one amino acid longer than the yeast actin. It is one of the most divergent actins sequenced to date, being only 75% homologous to yeast actin. Unlike the actin genes from most other organisms, it does not contain introns. The coding region contains TAA and TAG codons; the translation termination codon is TGA. Comparison of the amino acid sequence of the Tetrahymena actin with that of actins from other organisms suggests that TAG may code for glutamic acid. The gene is transcribed from multiple initiation sites between 57 and 98 nucleotides upstream of the translation start codon. The 5' flanking region is very A+T-rich and contains numerous "TATA-like" sequences upstream of the transcription start sites.

Journal Article↗