Cloning and sequencing of a jack bean urease-encoding cDNA.

A cDNA which encodes the entire amino acid (aa) sequence of the mature jack bean urease has been cloned in Escherichia coli from a library prepared from the mRNA of developing jack beans. It was necessary to use reverse transcriptase in the cDNA was obtained in the form of two contiguous DNA fragments, each of which was completely sequenced. The conceptual translation of the nt sequence gave an 840-aa sequence which was identical to the directly determined sequence except for one conservative aa substitution (Takashima et al., Eur. J. Biochem. 175 (1988) 151-165). These data constitute the first report on the cloning and sequence of the cDNA encoding a urease from any higher plant.

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon).

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance.

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

Mitochondrial sequence reveals high levels of gene flow between breeds of domestic sheep from Asia and Europe.

Sequence variation present within the mitochondrial genome was used to investigate genetic diversity within sheep breeds from Asia and Europe. Comparison of 2027 bp of sequence from 121 animals revealed 44 phylogenetically informative nucleotide positions and a single insertion/deletion. A total of 57 haplotypes were observed which formed two distinct clades. Type A haplotypes were found in breeds from Asia (India, Indonesia, Mongolia, and Tibet), while type B haplotypes were observed at the highest frequency in breeds sourced from Europe (nine breeds from Austria, Aland, Finland, Spain, and northwestern Russia). The distribution of haplotypes indicates sheep appear to have the weakest population structure and the highest rate of intercontinental dispersal of any domestic animal reported to date. Only 2.7% of the sequence variation observed was partitioned between continents, which is lower than both goat (approximately 10%) and cattle (approximately 50%). Diagnostic restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) tests which distinguish type A and B haplotypes were used to test an additional 223 animals from 17 breeds of European and Asian origin. A mixture of the two lineages was found in every breed except Suffolk and the Indian Garole, indicating introgression has played a major part during breed development and subsequent selection.

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua).

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 bp to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.