The keratin BIIIB gene family: isolation of cDNA clones and structure of a gene and a related pseudogene

The nucleotide sequence of cDNA clones encoding the three major BIIIB high-sulfur wool keratin proteins (BIIIB2, 3, and 4) and the structure of a BIIIB4 gene and a BIIIB3 pseudogene are reported. Although Southern blot analysis indicates that the BIIIB genes comprise a multigene family in the sheep genome, they are poorly represented in genomic DNA libraries. The family sequence homology of the coding region extends into the 5' and 3' untranslated regions and the near 5' flanking region of the BIIIB3 and 4 genes. These homologies suggest that the BIIIB3 and 4 genes represent the latest gene duplication event in the evolution of the BIIIB multigene family. Like the genes coding for other wool keratin matrix protein components, the BIIIB genes have the conserved 18-bp sequence immediately 5' to the initiation codon and also appear to lack introns.     

Complete nucleotide sequence of a functional HLA-DP beta gene and the region between the DP beta 1 and DP alpha 1 genes: comparison of the 5'' ends of HLA class II genes.

The complete nucleotide sequence of an HLA-DP beta 1 gene and part of the adjacent DP alpha 1 gene, up to and including the signal sequence exon, were determined. The sequence of the DP beta 1 gene identified it as the DPw4 allele. The six exons of the DP beta 1 gene spanned over 11,000 bp of sequence. The arrangement of the gene was broadly analogous to genes of other class II beta chains. The beta 1 exon was flanked by introns of over 4 kb. Comparisons with published sequences of cDNA clones indicated that an alternative splice junction, at the 3' end of the gene, is used in at least one allele. Variation in choice of splice junction indicates an additional mechanism for allelic variation in class II genes. The sequence also indicated that the DP beta 1 and DP alpha 1 genes are separated by only 2 kb at their 5' ends. Comparison of the 5' ends of the DP alpha 1 and beta 1 genes with other class II sequences, including the DZ alpha gene, showed conservation of several blocks of sequences thought to be involved in control of expression. Some areas of the introns were partially conserved in the DQ beta gene, and several other intron sequences were homologous to sequences found in other unrelated genes.     

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum,a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum, a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

Comparison of MHC genes among distantly related members of the genus Mus

The genomic content of class I and class II MHC DNA sequences in a variety of wild mice has been analyzed. The panel of mice includes members of three subgenera of the genus Mus. By genomic hybridization with the use of a variety of DNA probes, both class I and class II DNA sequences appear to be conserved in all of the species examined. However, the number of class I DNA sequences differs among the species. Furthermore, this variation appears to result from differential increases within subsets of class I genes. These data suggest that the class I multigene family is dynamic and changing over short periods of evolutionary time. In contrast, none of the class II genes appears to vary in copy number. More extensive polymorphism was noted amongst the class II beta genes than the alpha genes. Interestingly, the genomic sequence corresponding to E beta 2 is highly conserved, leading to the prediction that it is a genetically functional sequence.     

Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana

Plants synthesize a number of antimicrobial proteins in response to pathogen invasion and environmental stresses. These proteins include two classes of chitinases that have either basic or acidic isoelectric points and that are capable of degrading fungal cell wall chitin. We have cloned and determined the nucleotide sequence of the genes encoding the acidic and basic chitinases from Arabidopsis thaliana (L.) Heynh. Columbia wild type. Both chitinases are encoded by single copy genes that contain introns, a novel feature in chitinase genes. The basic chitinase has 73% amino acid sequence similarity to the basic chitinase from tobacco, and the acidic chitinase has 60% amino acid sequence similarity to the acidic chitinase from cucumber. Expression of the basic chitinase is organ-specific and age-dependent in Arabidopsis. A high constitutive level of expression was observed in roots with lower levels in leaves and flowering shoots. Exposure of plants to ethylene induced high levels of systemic expression of basic chitinase with expression increasing with plant age. Constitutive expression of basic chitinase was observed in roots of the ethylene insensitive mutant (etr) of Arabidopsis, demonstrating that root-specific expression is ethylene independent. Expression of the acidic chitinase gene was not observed in normal, untreated Arabidopsis plants or in plants treated with ethylene or salicylate. However, a transient expression assay indicated that the acidic chitinase promoter is active in Arabidopsis leaf tissue.     

Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize

The B-hordeins are the major group of prolamin storage proteins in barley (Hordeum vulgare L.) and they are encoded by a small multigene family that is expressed specifically in the developing endosperm. We report the complete nucleotide sequence of a clone of one B-hordein gene (pBHR184). The cloned gene contains no introns and belongs to the B1 sub-family of B-hordein genes. Comparison of the 5'-flanking sequences of pBHR184 with those of related S-rich prolamin genes from wheat shows that several short sequences within 600 bp upstream of the translation initiation codon are strongly conserved. A sequence that is conserved at around -300 bp in the S-rich prolamins is also conserved at similar locations in genes encoding the two major classes of maize prolamin (the Z19 and Z21 zeins) and appears to be unique to prolamin genes. We discuss the possible role of this '-300 element' in the control of gene expression in the developing cereal endosperm.     

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5 and 3 splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg²⁺- and ATP-dependent manner, and this activity was inhibited by 4-(9-acridinylamino)-3-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII ^ts yeast mutant.     

一种新的抗真菌几丁质酶基因的分离及其植物表达载体的构建

以西瓜尖镰孢菌诱导、提纯的豇豆抗真菌 I类几丁质酶 N端前 1 0个氨基酸序列测定的基础上 ,设计合成了引物 ,运用 PCR等分子生物学技术 ,从豇豆基因组中分离克隆了该特异几丁质酶成熟蛋白基因 ,测定分析了其全序列。该新基因全长 894bp,无内含子 ;具 Aat I、Aat II、Bgl I、Dpn I、Dpn II、Eco R II、Hae I、Hae II、Hae III、Hinf I、Hpa II、Mae II、Mae III、Nba I、Oxa I和 Sst IV酶切位点 43个 ;豇豆、Vigna unguiculata、菜豆、豌豆、烟草、小麦、水稻的同源性依次递减。扩增克隆了菜豆几丁质酶信号肽基因 ,并将其与豇豆几丁质酶成熟蛋白基因连接 ,再与 p BI1 2 1重组 ,成功构建了特异几丁质酶基因的植物表达载体 ,为进一步培育抗真菌病转基因西瓜新品种打下了坚实基础。     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed.