Cloning and sequencing of an apparently recombinant promoter for napin gene from Brassica campestris genomic library and its evolutionary significance.

From a genomic library of Brassica campestris (brown sarson cv. B54), we have cloned and sequenced about 2 kb of upstream regulatory region from one of the 2S albumin-coding gene family. The sequence has several seed-specific promoter motifs. A sequence alignment of the 5' flanking regions of the available Brassica 2S storage protein genes showed that our sequence is a double crossover recombinant product of the two members of the napin gene family. A possible explanation of this fact is that Brassica species evolved through gene duplication and recombination from a common ancestor with fewer number of chromosomes and genes.

Deduced amino acid sequence of 2S storage protein from Brassica species and their conserved structural features.

2S seed storage albumin coding regions from five Brassica species, namely Brassica campestris, B. oleracea, B. nigra, B. juncea, and B. carinata have been cloned by PCR amplification of genomic DNA using oligonucleotide primers and their nucleotide sequences have been determined. These sequences showed more than 85% homology amongst themselves and considerable homology with some other crucifer 2S protein coding sequences. The deduced amino acid sequences showed more homology due to some inconsequential mutations in codons without changing the amino acids. Computer analysis of the protein sequences for possible secondary structure revealed a high degree of conservation of hydrophilic and hydrophobic domains and the invariant positions of cysteine residues. Unrooted phylogenic tree based on the coding region of 2S albumin from different Brassica species cloned by us and published sequences from other Cruciferae indicated that these genes originated before the evolutionary divergence of different Brassica species and were conserved due to some stringent structural and functional features required for seed metabolism.

Bypassing successive cloning of two fragments by one step cloning using polymerase chain reaction.

The polymerase chain reaction (PCR) was used for bringing together two DNA fragments present in two different plasmids. This helped avoiding the difficulties of two successive subcloning in the same plasmid and uncertainities of obtaining rightly oriented constructs. Two different fragments of DNA present in different plasmid were digested with the same enzyme and then ligated. The ligation mixture was used for the PCR using two oligo primers; one was specific for the 5' end of the other fragment and the other one was for the 3' end of the other fragment. The desired amplified fragment was separated by gel electrophoresis, eluted and was cloned in plasmid pBluescript KS(+). The same procedure is also applicable for one step cloning of more than two fragments in desired orientation.

Organization and size class homogeneity of ribosomal RNA genes of catfish Heteropneustes fossilis.

The ribosomal RNA genes of catfish Heteropneustes fossilis Bloch were examined by Southern blot analysis of genomic DNA digested with restriction enzymes and probed with labelled catfish ribosomal RNA. The major repeat length is 12 kb and about 300 copies per haploid genome are tandemly arranged. The repeat lengths are homogeneous in size in different tissues and individuals. A restriction site polymorphism exists in some of the repeats.