Organization and expression of Drosophila tropomyosin genes

It has been shown (Jockusch &; Isenberg, 1981) that vinculin (130K protein) binds to actin and induces actin filaments to form bundles even at low ionic strength. Here, we present structural details on the vinculin molecule itself and on its interaction with actin. In negatively stained preparations, vinculin appeared as a globular protein with an average diameter of 85 Å. The ability of vinculin to form actin filament bundles was confirmed using shadowing techniques and gel analysis of sedimented material. Analysis of vinculin-induced paracrystals by optical diffraction and computer processing revealed their structural similarity to Mg-induced paracrystals. The lateral position of vinculin on surface-exposed actin filaments of such paracrystals was demonstrated directly in electron micrographs and indirectly by labelling vinculin with ferritin-coupled anti-vinculin F(ab′) fragments. Polymerization of actin in the presence of vinculin-coated polystyrene beads did not result in an “end-on” binding of filaments to the beads. Rather, actin bundles were laterally associated with the whole surface of the beads, from where they radiated in a star-like pattern. The growth of actin filaments onto myosin subfragment-I decorated, vinculin-incubated. fixed filament fragments was not inhibited, as was shown directly by electron microscopy and monitored viscometrically in a nucleation assay. These results suggest that in vivo at the site of an adhesion plaque vinculin may link actin filaments together into a suitable configuration to interact with the plasma membrane.     

Organization, structure and expression of murine interferon alpha genes.

Using a human interferon-alpha probe we have isolated recombinant phages containing murine interferon-alpha (Mu IFN-alpha) genes from a genomic library. One of these phages contained two complete Mu IFN-alpha genes and part of a third gene. The insert of a second phage held two IFN genes. This indicates that the Mu IFN-alpha genes are clustered in the genome as is the case for the analogous human genes. The nucleotide sequences of these 5 genes were determined. They show that the genes are all different, albeit highly homologous. The deduced amino acid sequences show that four of the five genes contain a putative glycosylation site. Three genes were transiently expressed in COS cells and they gave rise to protein products showing antiviral properties. The expression of the five Mu IFN-alpha genes and the Mu IFN-beta gene was studied in virus-induced mouse L cells. The individual mRNAs were visualized in a nuclease S1 experiment, using a specific probe for each gene. In RNA preparations from induced cells mRNAs for each of the five alpha genes and the beta gene were present. However, substantial differences in the amounts of the individual mRNAs were observed.     

Organization and expression of mouse Hox3 cluster genes

Summary The three yolk protein genes (yp) of Drosophila melanogaster are transcribed in a sex- and tissue-limited fashion. We have searched for cis-regulatory sequences in regions flanking yp1 and yp2 to identify the elements that confer female-specific expression in the fat body. One such 127 by element has previously been identified in this region. We show here the existence of two additional regions which confer female fat body-specific expression on an Adh reporter gene and on the native yp2 gene, respectively. This suggests some redundancy in the regulation of expression of the yp genes. Computer searches for putative binding sites for the DSX protein, which regulates sex-specific expression of the yp genes, revealed several such sites in our constructs. However, the significance of these is unclear since many such sites also occur in genes which one would not expect to be regulated in a sex-specific manner (e.g. Adh, Actin 5C). We suggest that DSX acts in concert with other proteins to mediate sex- and tissue-specific expression of the yp genes.     

Organization and expression of H1 histone and H1 replacement histone genes

The H1 family is the most divergent subgroup of the highly conserved class of histone proteins [Cole: Int J Pept Protein Res 30:433–449, 1987]. In several vertebrate species, the H1 complement comprises five or more subtypes, and tissue specific patterns of H1 histones have been described. The diversity of the H1 histone family raises questions about the functions of different H1 subtypes and about the differential control of expression of their genes. The expression of main type H1 genes is coordinated with DNA replication, whereas the regulation of synthesis of replacement H1 subtypes, such as H1° and H5, and the testis specific H1t appears to be more complex. The differential control of H1 gene expression is reflected in the chromosomal organization of the genes and in different promoter structures. This review concentrates on a comparison of the chromosomal organization of main type and replacement H1 histone genes and on the differential regulation of their expression. General structural and functional data, which apply to both H1 and core histone genes and which are covered by recent reviews, will not be discussed in detail.     

Organization and expression of genes involved in the biosynthesis of 987P fimbriae

Summary A chromosomal DNA segment encoding the biosynthesis of 987P fimbriae was isolated by cosmid-cloning and subsequent subcloning into pBR322. The 12 kb DNA segment expressed five polypeptides with apparent molecular weights of 81,000, 39,000, 28,500, 20,500, and 16,500, respectively. The location of the corresponding genes was determined by insertional mutagenesis using Tn5. The 20.5 K polypeptide was identified as the 987P fimbrial subunit by its reaction with specific anti-987P antibodies. The 81, 39, and 28.5 K polypeptides appeared to be accessory proteins involved in 987P production.     

Organization and expression of the SLC36 cluster of amino acid transporter genes

Three closely related genes encoding amino acid transport proteins are clustered on 5q32 in humans, and Chromosome (Chr) 11 in mice. The human SLC36A1 gene, which encodes the lysosomal amino acid transporter LYAAT1/PAT1, generates multiple alternative mRNAs, some of which encode truncated proteins. SLC36A1 is expressed in numerous tissues, whereas expression of SLC36A2, which encodes the glycine transporter tramdorin1/PAT2, is most abundant in kidney and muscle. Expression of a third gene, SLC36A3, is restricted to testis. Mouse Slc36a2 also is expressed in bone and fat tissue. Polymorphisms in human SLC36A2 exclude it as a candidate locus for a peripheral neuropathy that has been mapped to 5q31-33. SLC36A2 is a candidate gene for 5q-myelodysplastic syndrome, on the basis of its chromosomal location and its expression in bone.     

Preparation of a complementary DNA for leghaemoglobin and direct demonstration that leghaemoglobin is encoded by the soybean genome

In soybean root nodules, leghaemoglobin (Lb) accounts for 25--30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb in vitro, the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 9S-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 X 10(-3) and isolation of the hybridised cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

Organization,inheritance and expression of acetohydroxyacid synthase genes in the cotton allotetraploid Gossypium hirsutum

The acetohydroxyacid synthase (AHAS) gene family of the cotton AD allotetraploid Gossypium hirsutum has been cloned and characterized. We have identified six different AHAS genes from an analysis of genomic clones and Southern blots of genomic DNA. Four of the six genes are organized as tandem pairs, in which the genes are separated by only 2–3 kb. Conservation of restriction fragment length polymorphisms between G. hirsutum and A-genome and D-genome-containing diploid cottons was sufficient to assign the single genes in clones A5 and A19 to the A and D subgenomes, respectively. Each diploid genome has one tandem pair, but in these cases we could not make specific subgenomic assignments. DNA and deduced amino acid sequences were determined for the A5 and A19 genes, and an AHAS cDNA clone isolated from a leaflibrary. The sequence of the A19 gene matches that of the cDNA clone, while the A5 gene is 97.8% similar. The four genes comprising the tandem pairs are much less similar to the cDNA clone. The deduced amino acid sequences of the mature polypeptides encoded by the A5 and A19 genes are collinear with the housekeeping forms of AHAS from Arabidopsis thaliana, Nicotiana tabacum and Brassica napus. The constitutive expression of A5 and A19 was confirmed with RNase protection assays and northern blots. We conclude that these genes encode the main house-keeping froms of AHAS in G. hirsutum. Among the four AHAS genes comprising the two tandem pairs, at least two are functional. These genes exhibit either low-level constitutive expression (one or both of the downstream genes of each pair), or highly specific expression in reproductive tissue (one or both of the upstream genes of each pair). The AHAS gene family of G. hirsutum is more complex than that of other plants so far examined.