Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

Glutenins and gliadins are the main components of the wheat storage proteins and make up almost of the proteins found in gluten. Glutenins are polymeric pro-teins whose subunits are held together by in-ter-molecular disulphide bonds. They mainly consist o…     

Conservation and change in structural and 5′ flanking sequences of esterase 6 in siblingDrosophila species

Esterase 6 (Est-6/EST6) is the major β-carboxylesterase inD. melanogaster and its siblingsD. simulans andD. mauritiana. It is expressed in several tissues but its major site of expression is the sperm ejaculatory duct of the adult male. Although EST6 activity affects reproductive fitness, there are high levels of electrophoretic and activity polymorphism, at least withinD. melanogaster andD. simulans. Here we present the nucleotide sequences of anEst-6 allele and its flanking regions from each ofD. simulans andD. mauritiana and compare them with the publishedD. melanogaster sequences. As might be expected, replacement sites are significantly less divergent than exon silent sites in all comparisons, suggesting that selection is acting to maintain EST6 structure and function among the three species. Nevertheless, the ratio of the levels of replacement to silent site divergence is still much higher forEst-6 than for seven of ten other genes (including both isozyme-coding loci) for which comparable data have been published for these species. This is consistent with the high levels of EST6 electrophoretic polymorphism withinD. melanogaster andD. simulans and implies that selective constraints against amino acid change are relatively weak for EST6. By contrast, comparisons involving promotor sequences show that the level of divergence in the first 350bp 5′ of the gene is significantly lower than those for four of the six other loci for which comparable data have been published for these species. In particular, there are two perfectly conserved stretches (−1 to −158bp and −219 to −334bp) each over 100bp long included in this 350bp region. Thus the data suggest a relatively low level of selective constraint on the amino acid sequence of EST6 but a relatively high level of constraint on sequences affecting aspects of its expression.     

Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5′-flanking sequences

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30 000 M _r serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number L38482.     

Simple DNA sequences in homologous flanking regions near immunoglobulin VH genes: a role in gene interaction?

Five closely related immunoglobulin VH genes (subgroup II) were compared by sequencing of several kb of DNA. In three of the genes homology greater than 75% was found along an area of 4 kb that includes the coding region. The homology in flanking regions is only slightly lower than that in the coding sequences. Two other genes, which are located on the same EcoRI fragment, show high homology to the first three genes in the coding and immediately flanking regions. In more distant flanking regions no homology is found with the first three genes. This indicates that their evolutionary history differs from that of the other three genes. A region of simple DNA sequence composed of repetitive TCC and TCA elements was found at a distance of approximately 380 bp upstream from the initiator ATG of these VH genes. This region is the site where the two sets of genes abruptly start to diverge. The structure of the simple DNA sequence in the various VH genes suggests that it may be involved in gene interaction. We propose that both simple DNA sequences and homology in flanking regions serve a function in the correction of VH genes, which seem to be rather free to diverge and drift into pseudogenes. A correction mechanism may help this gene family to maintain its two major features, multiplicity and diversity.     

Comparison of effectiveness of 5′-regulatory sequences in transplastomic tobacco chloroplasts

The development of tools which ensure the desired level of transgene expression in plastids is a prerequisite for the effective utilization of these plant organelles for the deployment of bioactive proteins. High-level accumulation of target proteins is considered as a positive feature of transplastomic plants, but excessive accumulation of foreign proteins may have deleterious effects on host plants. On the other hand, expression at low levels can result in ineffective phenotypes. We compared the effectiveness of different 5′-regulatory sequences in driving the expression of a reporter gene, β-glucuronidase (uidA), in tobacco chloroplasts. To achieve varying expression levels, we have chosen heterologous 5′-regulatory sequences which either differ significantly from their homologous counterparts or depend on specific nuclear encoded factors. The Medicago truncatula psbA promoter/5′-UTR supported the highest levels of protein accumulation, surpassing the other tested sequences by two to three orders of magnitude. The heterologous regulatory sequence of Phaseolus vulgaris rbcL gene was as efficient in tobacco chloroplasts as the corresponding homologous promoter/5′-UTR. The Arabidopsis thaliana ndhF promoter/5′-UTR supported as high reporter activity levels as the rbcL 5′-sequences, whereas the effectiveness of A. thaliana psbN promoter/5′-UTR was three fold lower. The characterized regulatory sequences can be utilized to establish transplastomic lines with desirable levels of target protein accumulation. The ability to control transgene expression should be useful for achieving appropriate levels of protein accumulation and thereby avoid their negative impacts on host plant physiology.     

MUC genes: a superfamily of genes? Towards a functional classification of human apomucins

The MUC genes encode epithelial mucins. Eight different human genes have been well characterized, and two others identified more recently. Among them, a family of four genes, expressed in the respiratory and digestive tracts, is clustered to chromosome 11p15.5; and these genes encode gel-forming mucins which are structurally related to the superfamily of cystine-knot growth factors. A second group is composed of three independent genes encoding various isoforms of mucins including membrane-bound mucins associated to carcinomas. In this second group, MUC3 and MUC4 encode large apomucins containing EGF-like domains.     

Potential open reading frames in 5′-untranslated regions of eukaryotic mRNA

It is known that 5′-untranslated sequences of eukaryotic mRNA often contain AUG triplets, which can serve as translation initiation sites. It is assumed that such leader open reading frames can perform regulatory functions and code functionally active proteins; however, their characteristics have been studied insufficiently. In the article, the context organization of leader open reading frames of eukaryotic mRNA was considered. It was shown that their characteristics correlate with their position with respect to the protein-coding sequence, which may be related to the translation initiation efficiency.     

Cattle MHC nomenclature: is it possible to assign sequences to discrete class I genes?

The cattle major histocompatibility complex (MHC) region contains a variable number of classical class I genes encoding polymorphic molecules involved in antigen presentation. Six classical class I genes have been described, but assigning sequences to these genes has proved problematic. We propose a refinement of the existing nomenclature, which currently names the 97 known classical class I sequences in a single series. Phylogenetic analysis of the 3' portion of the coding region allows segregation of these into six groups; thus, we have prefixed existing names with the appropriate number. Although it is clear that some of these groups correspond to discrete genes, it is currently not possible to state definitively that all do. However, the main groupings are consistent, and in conjunction with other evidence, we feel it is now appropriate to rename the sequences accordingly. Segregation of sequences into groups in this way will facilitate ongoing research and future use of the cattle MHC section of the Immuno Polymorphism Database.