Saturated Molecular Map of the Rice Genome Based on an Interspecific Backcross Population

A molecular map has been constructed for the rice genome comprised of 726 markers (mainly restriction fragment length polymorphisms; RFLPs). The mapping population was derived from a backcross between cultivated rice, Oryza sativa, and its wild African relative, Oryza longistaminata. The very high level of polymorphism between these species, combined with the use of polymerase chain reaction-amplified cDNA libraries, contributed to mapping efficiency. A subset of the probes used in this study was previously used to construct an RFLP map derived from an inter subspecific cross, providing a basis for comparison of the two maps and of the relative mapping efficiencies in the two crosses. In addition to the previously described PstI genomic rice library, three cDNA libraries from rice (Oryza), oat (Avena) and barley (Hordeum) were used in this mapping project. Levels of polymorphism detected by each and the frequency of identifying heterologous sequences for use in rice mapping are discussed. Though strong reproductive barriers isolate O. sativa from O. longistaminata, the percentage of markers showing distorted segregation in this backcross population was not significantly different than that observed in an intraspecific F(2) population previously used for mapping. The map contains 1491 cM with an average interval size of 4.0 cM on the framework map, and 2.0 cM overall. A total of 238 markers from the previously described PstI genomic rice library, 250 markers from a cDNA library of rice (Oryza), 112 cDNA markers from oat (Avena), and 20 cDNA markers from a barley (Hordeum) library, two genomic clones from maize (Zea), 11 microsatellite markers, three telomere markers, eleven isozymes, 26 cloned genes, six RAPD, and 47 mutant phenotypes were used in this mapping project. Applications of a molecular map for plant improvement are discussed.     

Expression cloning of the murine erythropoietin receptor

Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.     

FISH physical mapping with barley BAC clones

Fluorescence in situ hybridization (FISH) is a useful technique for physical mapping of genes, markers, and other single- or low-copy sequences. Since clones containing less than 10 kb of single-copy DNA do not reliably produce detectable signals with current FISH techniques in plants, a bacterial artificial chromosome (BAC) partial library of barley was constructed and a FISH protocol for detecting unique sequences in barley BAC clones was developed. The library has a 95 kb average barley insert, representing about 20% of a barley genome. Two BAC clones containing hordein gene sequences were identified and partially characterized. FISH using these two BAC clones as probes showed specific hybridization signals near the end of the short arm of one pair of chromosomes. Restriction digests of these two BAC clones were compared with restriction patterns of genomic DNA; all fragments contained in the BAC clones corresponded to bands present in the genomic DNA, and the two BAC clones were not identical. The barley inserts contained in these two BAC clones were faithful copies of the genomic DNA. FISH with four BAC clones with inserts varying from 20 to 150 kb, showed distinct signals on paired chromatids. Physical mapping of single- or low-copy sequences in BAC clones by FISH will help to correlate the genetic and physical maps. FISH with BAC clones also provide an additional approach for saturating regions of interest with markers and for constructing contigs spanning those regions.     

Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Cytoplasmic RNA sequences complementary to cloned chick delta-crystallin cDNA show size heterogeneity.

Double-stranded complementary DNA (cDNA) sequences were prepared from day-old chick lens total polysomal RNA and inserted into the unique PstI restriction site of the plasmid pBR322. Colonies containing sequences complementary to abundant lens poly(A)-containing RNA sequences were identified by using lens 32P-labelled cDNA. Some of these clones have been characterized as containing delta-crystallin mRNA coding sequences by genomic DNA blot hybridization and RNA blot hybridizations. Hybridization of labelled DNA from such clones to RNA blots detected four size classes of delta-crystallin RNA sequences, although Southern blots indicated that there are probably only two delta-crystallin genes.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.

As part of the ongoing human and mouse genome projects, the aim of this study was to isolate novel, previously uncharacterized, genes from mouse testis. Two approaches were compared for their effectiveness in isolating novel genes: random, vs differential, complementary DNA (cDNA) cloning methods. In the differential approach, only the cDNA clones containing rare sequences (as determined by preliminary clone hybridization) are further analyzed; in the random approach, cDNA clones are isolated at random from the cDNA library. More than two hundred cDNA clones altogether were analyzed, using a PCR-mediated amplification and sequencing strategy. A comparison of these sequences to nucleic acid and protein sequence databases, revealed that 84% of the isolated rare cDNA clones represented new, previously uncharacterized mouse genes. In contrast, less than 63% of the cDNA clones isolated at random from cDNA libraries, contained novel genes. Thus, the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries can be markedly improved by focusing efforts on clones containing rare sequences.     

Cloning and partial characteristics of the barley D-hordein sequence]

A number of clones containing major endosperm-specifically transcribed gene copies were selected from a cDNA library developed on the basis of barley endosperm mRNA. Approx. 30% of the recombinant clones carried sequences homologous to mRNA of various cereal storage proteins. Some of them appeared to be related to cDNA clones of wheat and barley storage proteins. The typical insert length ranged from 0.3 to 1.7 kB. A couple of clones among them were selected which revealed positive hybridization with all probes used. The positive signals disappeared after stringent washing of the filters. The nucleotide sequences of two representatives of the group were determined and corresponding amino acid sequence deduced after subsequent computer analysis. The comparison with known cereal storage protein genes revealed relatively high homology level with the central part of wheat high molecular weight (HMW) glutenine subunit genes. The fact suggests the cloned gene to belong to barley D-hordein family.     

The human T cell receptor beta-chain gene complex contains at least 57 variable gene segments. Identification of six V beta genes in four new gene families.

The human TCR beta-chain gene complex includes at least 57 variable (V) gene segments, a number estimated using a combination of Southern blots of conventional and pulsed field gels, sequence analysis of cDNA clones, and from the analysis of genomic cosmid and phage clones. This number includes six TCR beta-chain V genes in four new families identified here by sequence analysis of clones derived from a human TCR beta-chain specific cDNA library. Comparison of the sequences of the new V beta genes with previously reported V beta sequences reveals predicted similarities but less than 75% nucleic acid identity that establishes them as new V beta families. One of the new V beta gene families includes three genes and the other three are single member families. Identification of these six new V beta genes falling into four V beta families brings the total number of transcribed human V beta families to 24 and makes it possible to refine the estimate of the total number of human TCR V beta genes to 57.     

Ly-49 multigene family. New members of a superfamily of type II membrane proteins with lectin-like domains

Ly-49 (YE1/48, A1) is a dimer protein expressed on subpopulations of murine NK cells. It is a member of a superfamily of type II transmembrane proteins containing carbohydrate recognition domains (CRD). In the mouse genome, the detection of multiple restriction fragments that cross-hybridize with Ly-49 cDNA probes suggests the presence of related genes. In this study, we have isolated several genomic clones encoding portions of CRD sequences highly homologous to the CRD of Ly-49. By using primers based on the consensus sequences of the genomic clones, expression of Ly-49-related genes was detected by the polymerase chain reaction in various organs, including lung, kidney, liver, spleen, and thymus. Two full-length cDNA clones that are highly homologous to the Ly-49 gene were subsequently isolated from a lung cDNA library. At the nucleotide level, the two clones are 72% and 80% identical to Ly-49 in their translated regions, but their sequences are different from those of the genomic clones characterized to date. The two cDNA clones potentially encode type II transmembrane proteins containing CRD that are very similar to Ly-49. These amino acid sequences are also homologous to other members of the superfamily of CRD-containing type II transmembrane proteins, including hepatic lectins and the low affinity IgER (CD23). The homology is most evident in the CRD but is also significant in other domains. These results demonstrate the existence of several functional genes that are highly related to Ly-49. These genes comprise a subfamily within the superfamily of type II transmembrane proteins containing CRD.     

Cloning and expression in Escherichia coli of cDNA for serine: pyruvate aminotransferase of rat liver

Cloned cDNAs for rat liver serine: pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme. Nineteen clones were isolated from 33 000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes. These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria. (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations. (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones. Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver. (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody. Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe. The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.     

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.     

Evaluation of a cDNA scanning method concerning the fidelity and efficiency of cDNA selection using the YAC CIC3B1-S region of Arabidopsis thaliana chromosome 5.

We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155 161). We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1-S DNA were concentrated. We isolated 545 cDNA clones from the sublibrary, and determined partial sequence of them to produce expressed sequence tags (ESTs) derived from the YAC region. In total, 74 nonredundant groups of cDNAs were obtained from 545 cDNA clones. Forty-seven percent of these EST clones had significant homology to functional proteins such as protein kinases, LON protease, nucleic acid binding protein and chloride channel protein. We compared the cDNA sequences isolated by the cDNA scanning method to the Arabidopsis genomic sequence corresponding to the YAC CIC3B1-S region, and found that 69% of the selected cDNAs are located in the region. We discuss the fidelity and efficiency of the cDNA scanning method for cloning region-specific cDNAs and its useful application in positional cloning.