K-Box Sequence of the Apetala1-Class MADS-Box Genes in Hexaploid Wheat

Direct amplification of genomic DNA from four wheat species produced DNA fragments corresponding to the K-box sequence of the apetala1/squamosa class of the MADS-box genes. Exons 3 to 5 were highly conserved within the tribe Triticeae and very similar to the apetala1 genes of darnel ryegrass, rice, and maize. Most of the variations observed were due to synonymous substitutions: the deduced amino acid sequences were 89–99% similar within the Triticeae and 88–94% within the entire family Poaceae. Introns 3 and 4 of the apetala1 class genes were similar in wheat and rye and differed from those in other MADS-box genes presently known.     

Putative MADS-Box Retropseudogenes in Rye (Secale L.)

The fragments of MADS-box genes belonging to the agamous and agamous-like structural classes were isolated by direct amplification of genomic DNA from annual rye (Secale cereale L.) and perennial rye (Secale montanum Guss.). The characterized fragments (deposited in the Genbank as the accession nos. AF332885–AF332887 and AF346894) comprise the complete sequences of exons 1 to 5 and lack corresponding introns. Their nearest homologs are the maize genes zag1 and zag5 (the Genbank accession nos. L18924 and L46398). One more agamous-like fragment isolated from annual rye (AF362364) is similar to theTaMADS12 wheat gene (AB007505). The fragment comprises exons 3–5 and contains a 105-bp insert between the exons 3 and 4; this insert does not resemble any MADS-box introns presently known. We assume that all these fragments of MADS-box genes are retropseudogenes.     

Homologs of the Genes for Receptor-Like Kinase Proteins Conferring Plant Resistance to Pathogens. Comparative Analysis of the Homologs of the Wheat Gene Cre3in the Triticeae

Direct genomic DNA amplification with the primers recognizing the NBS–kinase sequence of the wheat gene Cre3(Genbank accession AF052641) was used to obtain partial homologs of this gene in perennial and annual rye, wheat, and tall wheatgrass. The nucleotide sequences of the cloned fragments and their deduced amino acid sequences were compared to the already-known Cre3homologs in other wheat, aegilops, and barley genotypes. Within the tribe Triticeae, the extent of homology ranged from 86 to 94% for nucleotide sequences and from 74 to 96% for the deduced amino acid sequences, with the most variable region between Kin3 and PR3 conserved motifs.     

A new method for the construction of translationally coupled operons in a bacterial chromosome

A new method for the construction of translationally coupled operons in a bacterial chromosome was developed on the basis of the recombineering approach. The method includes the in vitro construction of an artificial operon with an efficiently translated proximal cistron, its insertion into the Escherichia coli chromosome, the modification of the operon via Red-driven insertion of a special “Junction” with an excisable selective marker into the intercistronic region of the initial operon, and the excision of the marker. The Junction structure was designed and tested. The Junction consists of three components. The first component is the E. coli rplC-rplD intercistronic region and serves for placing the TAA codon of the proximal gene in the SD sequence (TAAGGAG) of rplD. The second component is the Cm ^R gene flanked by λattL/R sites in such a fashion that the residual λattB site after λInt/Xis-driven excision of the marker does not contain termination codons in frame with ATG of rplD. The third component is the E. coli trpE-trpD intercistronic region which is added so that TGA of trpE acts a termination codon of the new open reading frame (ORF), while the overlapping (TGATG) ATG of trpD is in the position of the initiation codon of the distal gene of the original operon. The general design of the Junction provides the conversion of the original two-cistron operon into a three-cistron operon with translationally coupled genes, where the coupling of the artificial ORF (rplD’-λattB-’trpE) with the proximal gene is due to the rplC-rplD intercistronic region and its coupling with the distal gene is due to trpE-trpD. The strategy was experimentally implemented to construct an artificial operon P_tac-aroG4-serA5, where the expression the distal serA5 gene was optimized owing to translational coupling in a three-cistron operon.     

A Fragment of the rpoC2 Chloroplast RNA Polymerase Gene in Perennial and Annual Rye

A 1397-bp fragment corresponding to the rpoC2 chloroplast RNA polymerase gene was obtained by direct rye DNA amplification. Two rye species, Secale montanum Guss. and S. cereale L., did not practically differ in the structure of this DNA fragment (the nucleotide sequences were 99% identical). The corresponding nucleotide sequences in rye and wheat (Triticum aestivum L., Genbank accession no. AB027572) were 97–98% similar. The extent of the homology of various stretches of the rpoC2 rye gene with the corresponding sequences in maize and rice was 81–95%, whereas the deduced amino acid sequences of rpoC2 in rye, wheat, maize, and rice were considerably identical (96–97% of homology). The rye fragment of the rpoC2 gene differed from the corresponding sequences in three other grass species primarily by a short (49 bp) insert into the region of numerous short repeats corresponding to nucleotides 15750/15751, 28728/28729, and 27472/27473 in wheat, maize, and rice, respectively.