Soybean Genomic DNA Research Articles

Characterization and genomic organization of a highly expressed late nodulin gene subfamily in soybeans

A soybean nodulin cDNA clone (E41) hybrid-selected mRNA for three in vitro translation products with apparent molecular weights of 26 kDa, 25 kDa and 24 kDa. Based on Southern analysis of soybean genomic DNA, combined with mapping and sequencing of genomic clones, we identified four genes that are related to E41, one of which was identified to be the previously characterized N-20 gene. Our data indicate the linkage of three of the genes, of which one is a truncated version and suggest that they originated by gene duplication combined with deletion and conversion. The genes are highly expressed and we postulate that the sequence conservation in the 5' and 3' flanking regions of all four genes, has a functional role in their expression. Hybrid-selected translation products of E41 are not immunoprecipitable with antibody to the soluble fraction of nodules suggesting that they are membrane associated. The N-20 gene, which is a member of this gene subfamily, showed sequence similarity to four previously characterized nodulin genes and a phylogenetic tree is proposed based on the extent of sequence similarity.

Isolation and characterization of cDNA clones for plant cyclins.

We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency.

Characterization of a novel nodulin gene in soybean that shares sequence similarity to the gene for nodulin-24

A gene encoding for nodulin-16 (N-16) was isolated from a soybean genomic library. Nucleotide sequence analysis of the cDNA and the genomic clone of N-16 indicated that the coding region of this gene is 330 bp long and is interrupted by a single intron of 494 bp. The coding region of the N-16 gene shows a high degree of localized sequence similarity with the coding sequence of soybean nodulin-24 (N-24). Sequence similarity between the two genes is limited to the coding region of 90 bp in the first exon and the first 54 bp in the second exon of the N-16 gene which is repeated as the 2nd, 3rd, and 4th exons in the N-24 gene. The N-24 gene has been postulated to be a result of repeated duplication of an insertion element consisting of the 54 bp exon and the flanking intron sequences. In the absence of sequence similarity in the regions flanking the 54 bp sequence between the N-16 and N-24 genes, the N-16 gene does not appear to be the ancestral gene. Both N-16 and N-24 have a similar hydrophobic amino terminal end suggesting that N-16 like N-24 is targeted to the peribacteroid membrane. Southern analysis of soybean genomic DNA shows the presence of other related sequences to the N-16 gene, one of which is found to be closely linked to it. Analysis of the temporal accumulation of the N-16 transcripts during nodule development in effective and ineffective nodules suggests that N-16 and related genes might differ from leghemoglobin and some other late nodulin genes in their mechanism of regulation.

The isolation, characterization and sequence of two divergent ?-tubulin genes from soybean (Glycine max L.)

Two divergent β-tubulin genes (designated Sβ-1 and Sβ-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii β-tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different β-tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of β-tubulin genes (thus far undetected) exist in the soybean genome. The Sβ-1 and Sβ-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode β-tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with β-tubulins from several organisms showed that they are most homologous to Chlamydomonas β-tubulin (85-87%), with lesser degrees of homology to β-tubulins of vertebrate species (79-83%), Trypanosoma brucei (80-81%) and Saccharomyces cerevisiae (66-68%). The amino acid sequences of Sβ-1 and Sβ-2 are as divergent from each other as they are from the Chlamydomonas β-tubulin. The amino acids at the diverged positions in Sβ-2 are nearly all conservative substitutions while in Sβ-1, 18 of the 69 substitutions were non-conservative. Both soybean β-tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas β-tubulin genes. Codon usage in the two soybean β-tubulins is remarkably similar (D (2)=0.87), but differs from codon usage in other soybean genes.

Two soybean ribulose-1,5-bisphosphate carboxylase small subunit genes share extensive homology even in distant flanking sequences

Soybean contains a multigene family which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCss). A member of this gene family, SRS4, has been isolated from a soybean genomic DNA library. Its nucleotide sequence has been determined and compared to the sequence of SRS1, a previously characterized RuBPCss gene from soybean. Relevant regulatory sequences such as the PuPuCCAAT boxes, TATA box, the actual start of transcription and poly A addition sites are conserved between the two genes. Using a gene specific synthetic probe to the 3' flanking region the steady state mRNA levels of SRS4, like SRS1, are shown to be very high in light grown soybean seedlings and low in seedlings grown in darkness. SRS1 and SRS4 are very closely related, the three exons being 96%, 93% and 96.5% homologous in nucleotide sequence. The polypeptide sequences are nearly identical with only one amino acid change in each of the three exons encoding the 178 amino acid precursor polypeptide. The two introns are about 75% homologous and the flanking regions are more than 85% homologous (700 base pairs on the 5' end and 300 base pairs on the 3' end). Furthermore, hybridization studies between lambda clones containing the SRS1 and SRS4 genes reveal that a region of strong homology extends at least 4 kb on the 5' end and about 1.1 kb on the 3' end. We propose that these two genes may be alloalleles or homeologous alleles. This proposal is consistent with soybean having an allotetraploid origin, and would imply that the divergence of two ancient Papilionoidae species gave rise to these two genes.