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PubMed2004

A LIM protein, Hic-5, functions as a potential coactivator for Sp1.

Hic-5 is a LIM protein with striking similarity to paxillin, and shuttles between focal adhesions and the nucleus. Our previous study suggested that Hic-5 participates in the transcriptional control of several genes such as the c-fos and p21 genes. In the present study, we examined the function of Hic-5 in the nucleus using the transcriptional promoter region of the p21 gene. When localized to the nucleus, Hic-5 was found to transactivate the p21 promoter through two of five Sp1 sites in the region proximal to the TATA box. The Hic-5 effect was mediated by a transactivation domain of Sp1 and functional interaction with p300 through the LIM4 domain. Hic-5 was also shown to interact functionally and physically with Smad3 through the LIM domains and to potentiate p21 promoter activity together with Smad3 and Sp1. These properties were confirmed in an artificial system using GAL4-fusion protein. Thus, Hic-5 was suggested to have a potential function as a cofactor in the transcriptional complex that contains Sp1, playing a role in gene transcription in the nucleus as well as in integrin signaling at focal adhesion sites.

Animals↗
PubMed2002

Mechanisms regulating the expression, self-maintenance, and signaling-function of the bradykinin B2 and B1 receptors.

Bradykinin (BK) is a potent short-lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G-protein-coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Galphai and Galphaq G-protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg(10)-kallidin or desArg(9)-BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin-1 or tumor necrosis factor-alpha. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and self-maintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self-maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews (Regoli and Barabé, 1980, Pharmacol Rev 32:1-46; Regoli et al., 1990, J Cardiovasc Pharmacol 15(Suppl 6):S30-S38; Regoli et al., 1993, Can J Physiol Pharmacol 71:556-557; Marceau, 1995, Immunopharmacology 30:1-26; Regoli et al., 1998, Eur J Pharmacol 348:1-10).

Amino Acid Sequence↗
PubMed2004

Comparative genomic analysis of vertebrate Hox3 and Hox4 genes.

We used a comparative genomic approach to identify putative cis-acting regulatory sequences of the zebrafish hoxb3a and hoxb4a genes. We aligned genomic sequences spanning the clustered Hoxb1 to Hoxb5 genes from pufferfish, mice, and humans with the zebrafish hoxba and hoxbb cluster sequences. We identified multiple blocks of conserved sequences in non-coding regions within and surrounding the Hoxb3/b4 gene locus; a subset of these blocks are conserved in the zebrafish hoxbb cluster, despite loss of hoxb3/b4 genes. Overall, we find that the architecture of the Hoxb3/b4 loci and of the conserved sequence elements is very similar in teleosts and mammals. Our analyses also revealed two alternative transcripts of the zebrafish hoxb3a gene and an exon sequence unusually located 10 kb upstream of adjacent hoxb4a; an equivalent murine Hoxb3 exon has not yet been confirmed. We show that many of the Hoxb3/b4 conserved non-coding sequences correlate with functional neural enhancers previously described in the mouse. Further, within the conserved non-coding sequences we have identified binding sites for transcription factors, including Kreisler/Valentino, Krox20, Hox, and Pbx, some of which had not been previously described for the mouse. Finally, we demonstrate that the regulatory sequences of zebrafish hoxa3a are divergent with respect to the mouse ortholog Hoxa3, or the paralog hoxb3a. Despite limited conservation of regulatory sequences, zebrafish hoxa3a and hoxb3a genes share very similar expression profiles.

Animals↗
PubMed2005

Four types of Smad4 found in the common carp, Cyprinus carpio.

Smad4 is defined as the common-mediator Smad (Co-Smad) required for transducing signals for all transforming growth factor-beta (TGF-beta) superfamily members. In this study, we have isolated eight distinct Smad4 full-length cDNAs from the common carp (Cyprinus carpio). These cDNAs were classified into four types and each type consisted of two subtypes. The eight cDNAs encoded four distinct proteins ranging from 505aa to 568aa in size, with close similarities in the Mad homology 1 and 2 (MH1 and MH2, respectively), but with differences in the linker regions and the C-terminus as well as in the 5'- and 3'-untranslated regions. Genomic Southern blotting demonstrated the existence of at least six Smad4 gene loci in the carp genome, meaning that the multiple forms of the carp Smad4 cDNAs were not due to allelic variations. Reverse transcriptase polymerase chain reaction (RT-PCR)/Southern hybridizations showed different expression patterns among the four types of Smad4s. These results suggest that some of carp Smad4s have deviated from the original function of Smad4 through vertebrate evolution, and regulated the TGF-beta signaling pathway by changing the expression level in tissues.

Amino Acid Sequence↗
PubMed2000

Complement system of bony and cartilaginous fish.

Accumulating evidence indicates that the complement system experienced a discontinuous development at an early stage of vertebrate evolution. Invertebrates such as echinoderms and ascidians, and the most primitive extant vertebrates, the cyclostomes, seem to have a primitive complement system equipped only with the alternative and lectin pathways. In contrast, cartilaginous fish and higher vertebrates seem to have a modern complement system which has two additional pathways, namely the classical and lytic pathways. Recent molecular analyses of the complement system of bony and cartilaginous fish have not only confirmed the above conclusion, but also revealed a unique characteristic of the complement system of fish, where certain key component genes are duplicated. The complement system seems to play a more pivotal role in body defence in fish, whose adaptive immunity is considered to be at a relatively undeveloped state.

Amino Acid Sequence↗
PubMed1998

Pouch tissue and angiotensin peptide generation.

Myofibroblasts and their potential to generate angiotensin (Ang) II and transforming growth factor beta 1 (TGF-beta 1) at sites of infarction in the rat heart have been implicated in tissue repair. These cells likewise contribute to repair in a subcutaneous pouch model of fibrous tissue formation. Their appearance in pouch tissue coincides with high density ACE and Ang II receptor binding, suggesting a role for Ang II in tissue repair. Using pouch tissue studied at different time points of repair, the present study examined the expression of requisite mRNA for Ang peptide generation: angiotensinogen, Ao; an aspartyl protease, either cathepsin-D, Cat-D, or renin: and angiotensin converting enzyme, ACE, TGF-beta 1 and type I collagen mRNA expression was also addressed. Unlike pouch studied on day 2 and 4, at 7, 14 and 21 days, we found: (a) expression of Ao, Cat-D but not renin, ACE and TGF-beta 1 mRNA; (b) Ang I and Ang II peptides in pouch tissue and exudate; (c) the presence of Cat-D activity but no renin activity; (d) an increase in type I collagen mRNA with time; (e) upregulation of pouch tissue ACE mRNA expression by lisinopril treatment, whereas AT1 and AT2 receptor antagonists (losartan and PD 123177, respectively) downregulated the expression of mRNA for ACE, when compared to untreated controls; (f) downregulation of TGF-beta 1 mRNA expression by lisinopril and losartan compared to untreated controls; and (g) PD 123177 had no effect, whereas lisinopril and losartan treatment significantly (P < 0.05) reduced type I collagen mRNA expression. Thus, in this model of fibrous tissue formation, we found expression of component genes involved in Ang peptide (I and II) and TGF-beta 1 generation and Ang II upregulation of TGF-beta 1 expression, suggesting Ang II and/or TGF-beta 1 may upregulate type I collagen expression during tissue repair. Pharmacologic intervention studies with lisinopril or losartan indicate Ang II plays a role in the reciprocal regulation of ACE mRNA expression, which modulates Ang II levels at sites of repair.

Angiotensin I↗
PubMed1996

The complete nucleotide sequence of barley mild mosaic virus RNA1 and its relationship with other members of the Potyviridae.

The complete nucleotide sequence of RNA1 of a French barley mild mosaic bymovirus isolate has been determined. The molecule is composed of 7261 nucleotides and contains a single large open reading frame encoding a protein of 2258 amino acids with a calculated Mr of 256,375. Amino acid sequence comparison with poty- and rymoviruses reveals eight domains corresponding to the potyviral P3, 6K1, Cl, 6K2, Nla-VPg, Nla-Pro, Nlb and capsid proteins, respectively. Seven putative cleavage sites, similar to those mediated by potyvirus Nla proteinases, were identified. The presence of two, so far undescribed putative cleavage sites (6K2/Nla-VPg and Nla-VPg/Nla-Pro) in the polyproteins encoded by RNA1 of barley yellow mosaic virus and wheat spindle streak mosaic virus, was predicted. These data indicate that the bymovirus RNA1 fits the consensus potyviral genetic map downstream of the helper component gene.

Amino Acid Sequence↗
PubMed1975

Replication and expression of constructed plasmid chimeras in transformed Escherichia coli--a review.

EcoRI restriction-endonuclease-generated fragments of bacterial plasmids isolated from Staphylococcus aureus or Escherichia coli, or of amplified DNA coding for the 18S and 28S ribosomal RNA of Xenopus laevis, have been linked to the pSC101 plasmid replicon and introduced into E. coli by transformation. The constructed plasmid chimeras can be cloned as stable replicons in E. coli, where they synthesize RNA and/or protein products specified by their component genes.

Animals↗
PubMed2003

A new AOX homologous gene OsIM1 from rice (Oryza sativa L.) with an alternative splicing mechanism under salt stress.

A differentially expressed OsIM1 gene was isolated from rice salt-tolerant mutant M-20 by differential display. Sequence analysis revealed that the amino-acid sequence of OsIM1 showed 66% and 62% identity with PTOX from tomato ( Capsicum annuum) and AtIM from Arabidopsis, both of which encoded chloroplast-orientated terminal oxidase. Comparison of the nucleotide sequence of the OsIM1 cDNA with its genomic sequence revealed that OsIM1 genomic DNA contained nine exons and eight introns. A pseudo-transcript ( OsIM2), which probably resulted from the abnormal splicing of the OsIM1 pre-mRNA, was also identified. Southern-blot analysis showed that there existed only one copy of the OsIM1 gene in the rice genome. RFLP analysis located it on rice chromosome 3. The Northern blot revealed that OsIM1 was up-regulated by NaCl and ABA treatment. RT-PCR analysis indicated that OsIM1 and OsIM2 co-existed in the OsIM transcript pool, and the ratio of OsIM1/ OsIM2 was differentially regulated by salt stress in the salt-sensitive variety and the salt-tolerant varieties.

Alternative Splicing↗
PubMed2003

Organization of repetitive DNAs and the genomic regions carrying ribosomal RNA, cob, and atp9 genes in the cucurbit mitochondrial genomes.

Plants in the genus Cucumis (cucumber and melon) have the largest mitochondrial genomes known among all plants, due in part to the accumulation of repetitive DNAs of varying complexities. Recombination among these repetitive DNAs should produce highly rearranged mitochondrial genomes relative to the smaller mitochondrial genomes of related plants. We cloned and sequenced mitochondrial genomic regions near the rRNA, atp9 and cob genes from cucumber, melon, squash and watermelon (all members of the Cucurbitaceae family), and compared to the previously sequenced mitochondrial genomes of Arabidopsis thaliana and sugar beet to study the distribution and arrangement of coding and repetitive DNAs. Cucumber and melon had regions of concentrated repetitive DNAs spread throughout the sequenced regions; few repetitive DNAs were revealed in the mitochondrial genomes of A. thaliana, sugar beet, squash and watermelon. Recombination among these repetitive DNAs most likely produced unique arrangements of the rrn18 and rrn5 genes in the genus Cucumis. Cucumber mitochondrial DNA had more pockets of dispersed direct and inverted repeats than melon and the other plants, and we did not reveal repetitive sequences significantly contributing to mitochondrial genome expansion in both cucumber and melon.

Arabidopsis↗
PubMed2004

Gene content and density in banana ( Musa acuminata) as revealed by genomic sequencing of BAC clones.

The complete sequence of Musa acuminata bacterial artificial chromosome (BAC) clones is presented and, consequently, the first analysis of the banana genome organization. One clone (MuH9) is 82,723 bp long with an overall G+C content of 38.2%. Twelve putative protein-coding sequences were identified, representing a gene density of one per 6.9 kb, which is slightly less than that previously reported for Arabidopsis but similar to rice. One coding sequence was identified as a partial M. acuminata malate synthase, while the remaining sequences showed a similarity to predicted or hypothetical proteins identified in genome sequence data. A second BAC clone (MuG9) is 73,268 bp long with an overall G+C content of 38.5%. Only seven putative coding regions were discovered, representing a gene density of only one gene per 10.5 kb, which is strikingly lower than that of the first BAC. One coding sequence showed significant homology to the soybean ribonucleotide reductase (large subunit). A transition point between coding regions and repeated sequences was found at approximately 45 kb, separating the coding upstream BAC end from its downstream end that mainly contained transposon-like sequences and regions similar to known repetitive sequences of M. acuminata. This gene organization resembles Gramineae genome sequences, where genes are clustered in gene-rich regions separated by gene-poor DNA containing abundant transposons.

Base Composition↗
PubMed2004

Single nucleotide polymorphism, haplotype diversity and recombination in the Isa gene of barley.

The Isa gene from barley--an intronless gene expressed in maternal tissues of the seed--has a likely role in defence against pathogens. The protein product--bi-functional alpha-amylase/subtilisin inhibitor--inhibits the seed's own amylase in addition to the bacterial protease subtilisin and fungal xylanase. Sixteen barley genotypes were targeted to amplify and sequence the Isa gene region to detect sequence polymorphisms, since little is known about genetic diversity at this locus. A total of 80 single nucleotide polymorphisms (SNPs) and 23 indels were detected in 2,164 bp of sequence containing the Isa transcript, promoter and 3' non-transcribed region (overall one SNP per 27 bp and one indel per 94 bp), with eight sequence-based haplotypes distinguishable amongst the 16 varieties. Sequencing a polymorphic region in the promoter in an additional 27 barley genotypes increased the number of sequence-based haplotypes discovered to 11. However there is low haplotype diversity amongst the cultivated barley varieties sampled, with most varieties represented by a single haplotype. There was minor amino acid diversity in the protein, with five out of ten SNP sites in the coding region predicted to produce amino acid substitutions. SNP analysis indicated a history of recombination events--a minimum of seven based on the initial eight haplotypes from the whole sequenced region. Most of the recombination events occurred in the highly polymorphic regions, the 3' non-transcribed region and sequences flanking a microsatellite in the Isa promoter.

Base Sequence↗
PubMed2005

Development of a PCR-based marker utilizing a deletion mutation in the dihydroflavonol 4-reductase (DFR) gene responsible for the lack of anthocyanin production in yellow onions (Allium cepa).

Bulb color in onions (Allium cepa) is an important trait, but the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that inactivation of the dihydroflavonol 4-reductase (DFR) gene at the transcriptional level resulted in a lack of anthocyanin production in yellow onions. The objectives of the present study were the identification of the critical mutations in the DFR gene (DFR-A) and the development of a PCR-based marker for allelic selection. We report the isolation of two additional DFR homologs (DFR-B and DFR-C). No unique sequences were identified in either DFR homolog, even in the untranslated region (UTR). Both genes shared more than 95% nucleotide sequence identity with the DFR-A gene. To obtain a unique sequence from each gene, we isolated the promoter regions. Sequences of the DFR-A and DFR-B promoters differed completely from one another, except for an approximately 100-bp sequence adjacent to the 5'UTR. It was possible to specifically amplify only the DFR-A gene using primers designed to anneal to the unique promoter region. The sequences of yellow and red DFR-A alleles were the same except for a single base-pair change in the promoter and an approximately 800-bp deletion within the 3' region of the yellow DFR-A allele. This deletion was used to develop a co-dominant PCR-based marker that segregated perfectly with color phenotypes in the F2 population. These results indicate that a deletion mutation in the yellow DFR-A gene results in the lack of anthocyanin production in yellow onions.

Alcohol Oxidoreductases↗
PubMed2005

SNP identification and SNAP marker development for a GmNARK gene controlling supernodulation in soybean.

Supernodulation in soybean (Glycine max L. Merr.) is an important source of nitrogen supply to subterranean ecological systems. Single nucleotide-amplified polymorphism (SNAP) markers for supernodulation should allow rapid screening of the trait in early growth stages, without the need for inoculation and phenotyping. The gene GmNARK (Glycine max nodule autoregulation receptor kinase), controlling autoregulation of nodulation, was found to have a single nucleotide polymorphism (SNP) between the wild-type cultivar Sinpaldalkong 2 and its supernodulating mutant, SS2-2. Transversion of A to T at the 959-bp position of the GmNARK sequence results in a change of lysine (AAG) to a stop codon (TAG), thus terminating its translation in SS2-2. Based on the identified SNP in GmNARK, five primer pairs specific to each allele were designed using the WebSnaper program to develop a SNAP marker for supernodulation. One A-specific primer pair produced a band present in only Sinpaldalkong 2, while two T-specific pairs showed a band in only SS2-2. Both complementary PCRs, using each allele-specific primer pair were performed to genotype supernodulation against F2 progeny of Sinpaldalkong 2 x SS2-2. Among 28 individuals with the normal phenotype, eight individuals having only the A-allele-specific band were homozygous and normal, while 20 individuals were found to be heterozygous at the SNP having both A and T bands. Twelve supernodulating individuals showed only the band specific to the T allele. This SNAP marker for supernodulation could easily be analyzed through simple PCR and agarose gel electrophoresis. Therefore, use of this SNAP marker might be faster, cheaper, and more reproducible than using other genotyping methods, such as a cleaved amplified polymorphic sequence marker, which demand of restriction enzymes.

Base Sequence↗
PubMed2005

Comparative analysis of the D genome-encoded high-molecular weight subunits of glutenin.

Four genes encoding novel 1Dx-type high-molecular weight (HMW) subunits were amplified by polymerase chain reaction, two each from Aegilops tauschii and bread wheat Triticum aestivum. The two subunits from Ae. tauschii (1Dx2.1(t) and 1Dx2(t)) were both very similar in sequence to subunit 1Dx2 from bread wheat. In contrast, the two novel bread wheat subunits (1Dx2.2 and 1Dx2.2*) differed from subunit 1Dx2 in having different internally duplicated regions (of 132 and 186 amino acid, respectively) within their repetitive domains. These duplicated sequences were located adjacent to the regions from which they had been duplicated and had complete intact repeat motifs at each end. The implications of these results for HMW subunit evolution and wheat quality improvement are discussed.

Amino Acid Sequence↗
PubMed2005

Structural analysis of chloroplast DNA in Prunus (Rosaceae): evolution, genetic diversity and unequal mutations.

In order to understand the evolutionary aspects of the chloroplast DNA (cpDNA) structures in Rosaceous plants, a physical map of peach (Prunus persica cv. Hakuhou) cpDNA was constructed. Fourteen lambda phage clones which covered the entire sequence of the peach cpDNA were digested by restriction enzymes (SalI, XhoI, BamHI, SacI, and PstI) used singly or in combination. The molecular size of peach cpDNA was estimated to be about 152 kb. The gene order and contents were revealed to be equivalent to those of standard type of angiosperms by the localization of 31 genes on the physical map. Eighteen accessions from 14 Prunus species (P. persica, P. mira, P. davidiana, P. cerasis, P. cerasifera, P. domestica, P. insititia, P. spinosa, P. salicina, P. maritima, P. armeniaca, P. mume, P. tomentosa, P. zippeliana, and P. salicifolia) and one interspecific hybrid were used for the structural analysis of cpDNAs. Seventeen mutations (16 recognition site changes and one length mutation) were found in the cpDNA of these 18 accessions by RFLP analysis allowing a classification into 11 genome types. Although the base substitution rate in the recognition site (100p = 0.72) of cpDNA in Prunus was similar to that of other plants, i.e., Triticum-Aegilops, Brassica, and Pisum, it differed from Pyrus (100p = 0.15) in Rosaceae. Seven mutations including one length mutation were densely located within a region of about 9.1 kb which includes psbA and atpA in the left border of a large single-copy region of Prunus cpDNAs. The length mutation was detected only in P. persica and consisted of a 277 bp deletion which occurred in a spacer region between the trnS and trnG genes within the 9.1 kb region. Additional fragment length mutations (insertion/deletion), which were not detected by RFLP analysis, were revealed by PCR and sequence analyses in P. zippeliana and P. salicifolia. All of these length mutations occurred within the 9.1 kb region between psbA and atpA. This region could be an intra-molecular recombinational hotspot in Prunus species.

Base Sequence↗
PubMed2005

Identification of functional apple scab resistance gene promoters.

Apple scab (Venturia inaequalis) is one of the most damaging diseases affecting commercial apple production. Some wild Malus species possess resistance against apple scab. One gene, HcrVf2, from a cluster of three genes derived from the wild apple Malus floribunda clone 821, has recently been shown to confer resistance to apple scab when transferred into a scab-susceptible apple variety. For this proof-of-function experiment, the use of the 35S promoter from Cauliflower mosaic virus was reliable and appropriate. However, in order to reduce the amount of non-plant DNA in genetically modified apple to a minimum, with the aim of increasing genetically modified organism acceptability, these genes would ideally be regulated by their own promoters. In this study, sequences from the promoter region of the three members of the HcrVf gene family were compared. Promoter constructs containing progressive 5' deletions were prepared and used for functional analyses. Qualitative assessment confirmed promoter activity in apple. Quantitative promoter comparison was carried out in tobacco (Nicotiana glutinosa) and led to the identification of several promoter regions with different strengths from a basal level to half the strength of the 35S promoter from Cauliflower mosaic virus.

Ascomycota↗
PubMed2005

Identification of a large cluster of coiled coil-nucleotide binding site--leucine rich repeat-type genes from the Rps1 region containing Phytophthora resistance genes in soybean.

Fifteen Rps genes confer resistance against the oomycete pathogen Phytophthora sojae, which causes root and stem rot disease in soybean. We have isolated a disease resistance gene-like sequence from the genomic region containing Rps1-k. Four classes of cDNA of the sequence were isolated from etiolated hypocotyl tissues that express the Rps1-k-encoded Phytophthora resistance. Sequence analyses of a cDNA clone showed that the sequence is a member of the coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type of disease resistance genes. It showed 36% identity to the recently cloned soybean resistance gene Rpg1-b, which confers resistance against Pseudomonas syringae pv. glycinea, and 56% and 38% sequence identity to putative resistance gene sequences from lotus and Medicago truncatula, respectively. The soybean genome contains about 38 copies of the sequence. Most of these copies are clustered in approximately 600 kb of contiguous DNA of the Rps1-k region. We have identified a recombinant that carries both rps1-k- and Rps1-k-haplotype-specific allelomorphs of two Rps1-k-linked molecular markers. An unequal crossover event presumably led to duplication of alleles for these two physically linked molecular markers. We hypothesize that the unequal crossing over was one of the mechanisms involved in tandem duplication of CC-NBS-LRR sequences in the Rps1-k region.

Amino Acid Sequence↗