Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2–76 (or 75) of SAA2α and the other corresponded to positions 2–76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1α has valine and alanine and SAA1β has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA1^52,57Ala.     

Primary structure of a protein isolated from reef shark (Carcharhinus springeri) cartilage that is similar to the mammalian C-type lectin homolog, tetranectin.

During the course of characterization of low molecular weight proteins in cartilage, we have isolated a protein from reef shark (Carcharhinus springeri) cartilage that bears a striking resemblance to the tetranectin monomer originally described by Clemmensen et al. (1986, Eur. J. Biochem. 156, 327-333). The protein was isolated by extraction of neural arch cartilage with 4 M guanidine hydrochloride, dialysis of the extract to bring the guanidine to 0.4 M (reassociating proteoglycan aggregates), followed by cesium chloride density gradient removal of the proteoglycans. The amino acid sequence had 166 amino acids and a calculated molecular weight of 18,430. The shark protein was 45% identical to human tetranectin, indicating that it was in the family of mammalian C-type lectins and that it was likely to be a shark analog of human tetranectin. The function of tetranectin is unknown; it was originally isolated by virtue of its affinity for the kringle-4 domain of plasminogen. Sequence comparison of human tetranectin and the shark-derived protein gives clues to potentially important regions of the molecule.