RESUMO
Photosynthetic complexes in the thylakoid membrane of plant leaves primarily function as energy-harvesting machinery during the growth period. However, leaves undergo developmental and functional transitions along aging and, at the senescence stage, these complexes become major sources for nutrients to be remobilized to other organs such as developing seeds. Here, we investigated age-dependent changes in the functions and compositions of photosynthetic complexes during natural leaf senescence in Arabidopsis thaliana. We found that Chl a/b ratios decreased during the natural leaf senescence along with decrease of the total chlorophyll content. The photosynthetic parameters measured by the chlorophyll fluorescence, photochemical efficiency (F v/F m) of photosystem II, non-photochemical quenching, and the electron transfer rate, showed a differential decline in the senescing part of the leaves. The CO2 assimilation rate and the activity of PSI activity measured from whole senescing leaves remained relatively intact until 28 days of leaf age but declined sharply thereafter. Examination of the behaviors of the individual components in the photosynthetic complex showed that the components on the whole are decreased, but again showed differential decline during leaf senescence. Notably, D1, a PSII reaction center protein, was almost not present but PsaA/B, a PSI reaction center protein is still remained at the senescence stage. Taken together, our results indicate that the compositions and structures of the photosynthetic complexes are differentially utilized at different stages of leaf, but the most dramatic change was observed at the senescence stage, possibly to comply with the physiological states of the senescence process.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Tilacoides/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Fluorescência , Complexos de Proteínas Captadores de Luz/metabolismo , Processos Fotoquímicos , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Fatores de TempoRESUMO
Recent publications have revealed that the evolution of phosphosites is influenced by the local protein structures and whether the phosphosites have characterized functions or not. With knowledge of the wide functional range of phosphorylation, we attempted to clarify whether the evolutionary conservation of phosphosites is different among distinct functional modules. We grouped the phosphosites in the human genome into the modules according to the functional categories of KEGG (Kyoto Encyclopedia of Genes and Genomes) and investigated their evolutionary conservation in vertebrate genomes from mouse to zebrafish. We have found that the phosphosites in the vertebrate-specific functional modules (VFMs), such as cellular signaling processes and responses to stimuli, are evolutionarily more conserved than those in the basic functional modules (BFMs), such as metabolic and genetic processes. The phosphosites in the VFMs are also significantly more conserved than their flanking regions, whereas those in the BFMs are not. These results hold for both serine/threonine and tyrosine residues, although the fraction of phosphorylated tyrosine residues is increased in the VFMs. Moreover, the difference in the evolutionary conservation of the phosphosites between the VFMs and BFMs could not be explained by the difference in the local protein structures. There is also a higher fraction of phosphosites with known functions in the VFMs than BFMs. Based on these findings, we have concluded that protein phosphorylation may play more dominant roles for the VFMs than BFMs during the vertebrate evolution. As phosphorylation is a quite rapid biological reaction, the VFMs that quickly respond to outer stimuli and inner signals might heavily depend on this regulatory mechanism. Our results imply that phosphorylation may have an essential role in the evolution of vertebrates.
Assuntos
Evolução Molecular , Redes Reguladoras de Genes/fisiologia , Fosfoproteínas/metabolismo , Vertebrados/genética , Animais , Sequência Conservada , Genoma , Humanos , Fosfoproteínas/classificação , Fosfoproteínas/genética , Fosforilação , Proteômica , Serina/metabolismo , Relação Estrutura-Atividade , Treonina/metabolismo , Tirosina/metabolismo , Vertebrados/metabolismoRESUMO
BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide and this drives us to further understand their biology so that effective control measures can be developed. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina technology to sequence the small RNA transcriptomes derived from various life stages and selected organs of R. microplus. RESULTS: To discover and profile cattle tick miRNAs we employed two complementary approaches, one aiming to find evolutionary conserved miRNAs and another focused on the discovery of novel cattle-tick specific miRNAs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. CONCLUSIONS: Anciently acquired miRNAs in the R. microplus lineage not only tend to accumulate the least amount of nucleotide substitutions as compared to those recently acquired miRNAs, but also show ubiquitous expression profiles through out tick life stages and organs contrasting with the restricted expression profiles of novel tick-specific miRNAs.
Assuntos
Evolução Molecular , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Rhipicephalus/genética , Animais , Sequência de Bases , Bovinos , Drosophila/genética , Feminino , Perfilação da Expressão Gênica , Larva/genética , Masculino , MicroRNAs/genética , Família Multigênica , Óvulo/metabolismo , Precursores de RNA/genética , Rhipicephalus/crescimento & desenvolvimentoRESUMO
DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) collected and released 2 368 110 entries or 1 415 106 598 bases in the period from July 2007 to June 2008. The releases in this period include genome scale data of Bombyx mori, Oryzas latipes, Drosophila and Lotus japonicus. In addition, from this year we collected and released trace archive data in collaboration with National Center for Biotechnology Information (NCBI). The first release contains those of O. latipes and bacterial meta genomes in human gut. To cope with the current progress of sequencing technology, we also accepted and released more than 100 million of short reads of parasitic protozoa and their hosts that were produced by using a Solexa sequencer.
Assuntos
Bases de Dados de Ácidos Nucleicos , Análise de Sequência de DNA/tendências , Animais , Genômica , HumanosRESUMO
The Genomes TO Protein Structures and Functions (GTOP) database (http://spock.genes.nig.ac.jp/~genome/gtop.html) freely provides an extensive collection of information on protein structures and functions obtained by application of various computational tools to the amino acid sequences of entirely sequenced genomes. GTOP contains annotations of 3D structures, protein families, functions, and other useful data of a protein of interest in user-friendly ways to give a deep insight into the protein structure. From the initial 1999 version, GTOP has been continually updated to reap the fruits of genome projects and augmented to supply novel information, in particular intrinsically disordered regions. As intrinsically disordered regions constitute a considerable fraction of proteins and often play crucial roles especially in eukaryotes, their assignments give important additional clues to the functionality of proteins. Additionally, we have incorporated the following features into GTOP: a platform independent structural viewer, results of HMM searches against SCOP and Pfam, secondary structure predictions, color display of exon boundaries in eukaryotic proteins, assignments of gene ontology terms, search tools, and master files.
Assuntos
Bases de Dados de Proteínas , Conformação Proteica , Proteínas/genética , Éxons , Genômica , Proteínas/química , Proteínas/fisiologia , Análise de Sequência de Proteína , SoftwareRESUMO
The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genoma de Planta , Oryza/genética , Genes de Plantas , Genômica , Internet , MicroRNAs/genética , RNA Interferente Pequeno/genética , Interface Usuário-ComputadorRESUMO
The Center for Information Biology and DNA Data Bank of Japan (CIB-DDBJ) has operated biological databases since 1987 in collaboration with NCBI and EBI. As one of the three major public databases, CIB-DDBJ has run four primary databases DDBJ, CIBEX, DDBJ Trace Archive (DTA), and DDBJ Read Archive (DRA) to collect, archive, and provide various kinds of biological data. As the massively parallel new sequencing platforms are increasingly in use, huge amounts of the raw data have been produced. To archive these raw data, we at CIB-DDBJ began operating a new repository, the DDBJ Read Archive (DRA). To accommodate efficiently the processed data as well, we have developed a new pipeline, the DDBJ Read Annotation Pipeline that deals with both data submission and analysis. For data produced by the next generation platforms, the three archives DRA, DDBJ, and CIBEX, which are interconnected by the pipeline, collect the raw, processed sequence, and quantitative data, respectively. The public biological databases at CIB-DDBJ, EBI, and NCBI will together construct world-wide archives for biological data by data sharing to accelerate research in life sciences in the era of next generation sequencing technologies.
Assuntos
Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Análise de Sequência de DNA/estatística & dados numéricos , Biologia Computacional , Bases de Dados de Ácidos Nucleicos/tendências , Japão , Modelos Estatísticos , Análise de Sequência de DNA/tendênciasRESUMO
SUMMARY: BioCaster is an ontology-based text mining system for detecting and tracking the distribution of infectious disease outbreaks from linguistic signals on the Web. The system continuously analyzes documents reported from over 1700 RSS feeds, classifies them for topical relevance and plots them onto a Google map using geocoded information. The background knowledge for bridging the gap between Layman's terms and formal-coding systems is contained in the freely available BioCaster ontology which includes information in eight languages focused on the epidemiological role of pathogens as well as geographical locations with their latitudes/longitudes. The system consists of four main stages: topic classification, named entity recognition (NER), disease/location detection and event recognition. Higher order event analysis is used to detect more precisely specified warning signals that can then be notified to registered users via email alerts. Evaluation of the system for topic recognition and entity identification is conducted on a gold standard corpus of annotated news articles. AVAILABILITY: The BioCaster map and ontology are freely available via a web portal at http://www.biocaster.org.
Assuntos
Armazenamento e Recuperação da Informação/métodos , Vigilância da População , Software , Humanos , Internet , Saúde PúblicaRESUMO
DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) newly collected and released 12,927,184 entries or 13,787,688,598 bases in the period from July 2005 to June 2006. The released data contain honeybee expressed sequence tags (ESTs), re-examined and re-annotated complete genome data of Escherichia coli K-12 W3110, medaka WGS and human MGA. We also systematically evaluated and classified the genes in the complete bacterial genomes submitted to the International Nucleotide Sequence Database Collaboration (INSDC, http://insdc.org) that is composed of DDBJ, EMBL Bank and GenBank. The examination and classification selected 557,000 genes as reliable ones among all the bacterial genes predicted by us.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genes Bacterianos , Animais , Classificação , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Estudos de Avaliação como Assunto , Genoma Bacteriano , Humanos , Internet , Fases de Leitura AbertaRESUMO
AIM: Currently, approximately 44 000 hepatitis C virus (HCV), 11 000 hepatitis B virus (HBV), and 1600 hepatitis E virus (HEV) sequences are available at the International Nucleotide Sequence Database Collaboration (INSDC, previously known as DDBJ/EMBL/GenBank), and the number of these virus sequences is growing rapidly. However, since INDSC is not specialized to hepatitis viruses, it is difficult to retrieve information of virological or clinical interests from it. Thus, it is quite worthwhile to construct a specialized database for the hepatitis virus sequences and to make it accessible to researchers worldwide. METHODS: We developed a WWW-based database hepatitis virus database (HVDB), which contains all the HCV, HBV, and HEV sequences available at INSDC. In the HVDB, all piece sequences obtained from INSDC are arranged to the genomesequence of each virus. Also given in the database are the phylogenetic relationships of each locus on the genome among variants for each virus. RESULTS: Users of the database can easily retrieve entries (sequences with annotations) of the specific genotype by referring to the phylogenetic relationships or those of specific loci by referring to the genome map information. HVDB provides users with a tool for phylogenetic analysis that can be used in combination with the data retrieval tools. CONCLUSION: The latest release is publicly accessible at the HVDB website: http://s2as02.genes.nig.ac.jp.
RESUMO
Lactobacillus gasseri ATCC33323(T) has seven putative phospho-beta-glycosidase genes. Using column chromatography, we found that this strain cultured in lactose medium expresses five phospho-beta-glycosidases (LacG1, LacG2, Pbg1, Pbg2, and Pbg3), where these gene expressions can be suppressed by glucose. To our knowledge, this is the first report indicating that five glycosidases are induced from a single bacterial strain using a single carbon source, lactose.
Assuntos
Lactobacillus/enzimologia , Lactose/farmacologia , beta-Galactosidase/isolamento & purificação , Cromatografia , Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Lactobacillus/metabolismo , Técnicas Microbiológicas , Fosfoproteínas/isolamento & purificaçãoRESUMO
In the past year, DDBJ (http://www.ddbj.nig.ac.jp) collected and released 1,956,826 entries or 1,741,313,111 bases. The released data include approximately 90,000 ESTs and cDNAs of Macaca fascicularis, and 280 million bases of mouse GSS. In addition to the data collection, we have indexed the submitted data to the International Nucleotide Sequence Database Collaboration (INSDC, http://www.insdc.org) to classify the entries into research projects behind data submissions. They are expected to be useful to the data submitters and users for enhancing the data submission, retrieval and systematic data analyses at INSDC. The results of indexing also allow one to grasp research projects in life sciences that promoted and produced the DNA sequences submitted to INSDC.
Assuntos
Bases de Dados de Ácidos Nucleicos , Indexação e Redação de Resumos , Animais , Sequência de Bases , DNA/química , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Geografia , Humanos , Internet , Macaca fascicularis , Camundongos , Pesquisa , Interface Usuário-ComputadorRESUMO
A large number of complete microorganism genomes has been sequenced and submitted to the public database and then incorporated into our complete genome database, Genome Information Broker (GIB, http://gib.genes.nig.ac.jp/). However, when comparative genomics is carried out, researchers must be aware that there are protein-coding genes not confirmed by homology or motif search and that reliable protein-coding genes are missing. Therefore, we developed a protocol (Gene Trek in Prokaryote Space, GTPS) for finding possible protein-coding genes in bacterial genomes. GTPS assigns a degree of reliability to predicted protein-coding genes. We first systematically applied the protocol to the complete genomes of all 123 bacterial species and strains that were publicly available as of July 2003, and then to those of 183 species and strains available as of September 2004. We found a number of incorrect genes and several new ones in the genome data in question. We also found a way to estimate the total number of orthologous genes in the bacterial world.
Assuntos
Bactérias/classificação , Genes Bacterianos , Genética Microbiana , Genoma Bacteriano , Bactérias/genética , Biologia Computacional , DNA Bacteriano/genética , Sistemas de Gerenciamento de Base de Dados , Fases de Leitura Aberta , Células ProcarióticasRESUMO
Fundamental biological processes can now be studied by applying the full range of OMICS technologies (genomics, transcriptomics, proteomics, metabolomics, and beyond) to the same biological sample. Clearly, it would be desirable if the concept of sample were shared among these technologies, especially as up until the time a biological sample is prepared for use in a specific OMICS assay, its description is inherently technology independent. Sharing a common informatic representation would encourage data sharing (rather than data replication), thereby reducing redundant data capture and the potential for error. This would result in a significant degree of harmonization across different OMICS data standardization activities, a task that is critical if we are to integrate data from these different data sources. Here, we review the current concept of sample in OMICS technologies as it is being dealt with by different OMICS standardization initiatives and discuss the special role that the newly formed Genomic Standards Consortium (GSC) might have to play in this domain.
Assuntos
Bases de Dados de Ácidos Nucleicos/normas , Genoma Humano , Genoma , Genômica/normas , Proteoma/genética , Proteômica/normas , Animais , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/normasRESUMO
The Third Party Annotation (TPA) project collects and presents high-quality annotation of nucleotide sequence. Annotation is submitted by researchers who have not themselves generated novel nucleotide sequence. In its first few years, the resource has proven to be popular with submitters from a range of biological research areas. Central to the project is the requirement for high-quality data, resulting from experimental and inferred analysis discussed in peer-reviewed publications. The data are divided into two tiers: those with experimental evidence and those with inferential evidence. Standards for TPA are detailed and illustrated with the aid of case studies.
Assuntos
Bases de Dados de Ácidos Nucleicos/normas , Genômica/normas , Animais , Coleta de Dados/normas , HumanosRESUMO
Orthologs are widely used for phylogenetic analysis of species; however, identifying genuine orthologs among distantly related species is challenging, because genes obtained through horizontal gene transfer (HGT) and out-paralogs derived from gene duplication before speciation are often present among the predicted orthologs. We developed a program, "Ortholog-Finder," to obtain ortholog data sets for performing phylogenetic analysis by using all open-reading frame data of species. The program includes five processes for minimizing the effects of HGT and out-paralogs in phylogeny construction: 1) HGT filtering: Genes derived from HGT could be detected and deleted from the initial sequence data set by examining their base compositions. 2) Out-paralog filtering: Out-paralogs are detected and deleted from the data set based on sequence similarity. 3) Classification of phylogenetic trees: Phylogenetic trees generated for ortholog candidates are classified as monophyletic or polyphyletic trees. 4) Tree splitting: Polyphyletic trees are bisected to obtain monophyletic trees and remove HGT genes and out-paralogs. 5) Threshold changing: Out-paralogs are further excluded from the data set based on the difference in the similarity scores of genuine orthologs and out-paralogs. We examined how out-paralogs and HGTs affected phylogenetic trees constructed for species based on ortholog data sets obtained by Ortholog-Finder with the use of simulation data, and we determined the effects of confounding factors. We then used Ortholog-Finder in phylogeny construction for 12 Gram-positive bacteria from two phyla and validated each node of the constructed tree by comparison with individually constructed ortholog trees.
Assuntos
Transferência Genética Horizontal , Fases de Leitura Aberta , Análise de Sequência de DNA/métodos , Software , Genoma Bacteriano , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , FilogeniaRESUMO
We developed a reliability index named SRED (Spot Reliability Evaluation Score for DNA microarrays) that represents the probability that the calibrated gene expression level from a DNA microarray would be less than a factor of 2 different from that of quantitative real-time polymerase chain reaction assays whose dynamic quantification range is treated statistically to be similar to that of the DNA microarray. To define the SRED score, two parameters, the reproducibility of measurement value and the relative expression value were selected from nine candidate parameters. The SRED score supplies the probability that the expression level in each spot of a microarray is less than a certain-fold different compared to other expression profiling data, such as QRT-PCR. This score was applied to approximately 1,500,000 points of the expression profile in the RIKEN Expression Array Database.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Algoritmos , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Cerebelo/metabolismo , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Fator 3-beta Nuclear de Hepatócito , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , RNA/genética , RNA/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Fatores de Tempo , Fatores de Transcrição/genética , TransfecçãoRESUMO
While highly differentiated and long-conserved sex chromosomes such as XY and ZW chromosomes are observed, respectively, in mammalian and avian species, no counterparts to such chromosomes were observed in fish until we reported in the previous study that well-conserved and highly differentiated ZW sex chromosomes existed in the family of Synodontidae. Then, the problem was if the evolutionary history of the fish ZW chromosomes was long enough to be comparable to the mammalian and avian counterparts. To tackle the problem, we had to extend our finding of the fish sex chromosomes further than a family alone. For this purpose, we chose Aulopus japonicus that belonged to one of the related families to Synodontidae. Our cytogenetic and fluorescence in situ hybridization (FISH) analyses have clearly demonstrated that A. japonicus also has ZW chromosomes. We have also found that 5S rDNA clusters are located on the Z and W chromosomes in this species. Using nontranscribed intergenic sequences in the 5S rDNA clusters as PCR primers, we successfully amplified a 6-kb-long female-specific sequence on the W chromosome. The 6-kb-long sequence contained one transposable element and two tRNA sequences. The function of the sequence remains to be studied. Our Southern blot analysis confirmed that the 6-kb sequence was located only on the W chromosome.Therefore, it is now said that highly differentiated ZW chromosomes have been conserved over two fish families. As these families were reported to have been diverged 30-60 million years ago, the fish ZW chromosomes have an evolutionary history corresponding to the history of the families. This is perhaps the first case that fish sex chromosomes are shown to have such a long evolutionary lineage.
Assuntos
Evolução Molecular , Peixes/genética , Cromossomos Sexuais/genética , Animais , Sequência de Bases , Feminino , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , RNA Ribossômico 5S/genética , Fatores SexuaisRESUMO
The Lake Victoria Cichlid fishes have diverged very rapidly. The estimated 500 species inhabiting the lake are believed to have arisen within the last 14,000 years. The fishes' jaws and teeth have diverged markedly to adapt to different feeding behaviors and environments. To examine how the genomes of these fishes differentiated during speciation, we performed comparative analysis of expressed sequenced tag (EST) sequences. We constructed cDNA libraries derived only from the jaw portions of two cichlid species endemic to Lake Victoria. We sequenced 17,280 cDNA clones from Haplochromis chilotes and 9600 cDNA clones from Haplochromis sp. "Redtailsheller" and obtained 543 different genes common to both species. Of these genes, 441 were essentially identical between species and 102 contained base replacements in their open reading frame (ORF) or untranslated (UTR) regions. Comparative analysis of 71 selected sequences has revealed that while the degree of polymorphism is 0.0054/site for H. chilotes and 0.0047/site for H. sp. "Redtailsheller", genetic distance between the two species is 0.0031/site. The genetic distance particularly indicates that the two species diverged about 890,000 years ago.
Assuntos
Ciclídeos/genética , Etiquetas de Sequências Expressas , Fases de Leitura Aberta , Análise de Sequência de DNA , África Oriental , Animais , Evolução Biológica , Ciclídeos/classificação , Biblioteca Gênica , Genes Homeobox , Desenvolvimento Maxilofacial/genética , Polimorfismo GenéticoRESUMO
We have developed a novel method for cloning gene family members by using a polymerase chain reaction technique. The method is based on the amplification of a broad range of homologous genes in combination with the specific inhibition of already cloned genes. To accomplish this, we designed degenerate primers to highly conserved regions among the gene family members, and inhibitory primers to the divergent region at the 3'-margin of each degenerate primer. The 5'-end of the inhibitory primer, the 3'-end of which was aminated, had 3-4 bases overlapping the 3'-end of the degenerate primer. The potential of this method was demonstrated by the successful cloning of a novel member of the yeast MKC7/YAP3 gene family homologue from a filamentous fungus, Aspergillus oryzae, by inhibiting amplification of an already cloned homologue, opsB.