Analysis of the expression and function of the small heat shock protein gene, hsp27, in Xenopus laevis embryos

In previous studies, the only small HSPs that have been studied in Xenopus laevis are members of the HSP30 family. We now report the analysis of Xenopus HSP27, a homolog of the human small HSP, HSP27. To date the presence of both hsp30 and hsp27 genes has been demonstrated only in minnow and chicken. Xenopus HSP27 cDNA encodes a 213 aa protein that contains an alpha-crystallin domain as well as a polar C-terminal extension. Xenopus HSP27 shares 71% identity with chicken HSP24 but only 19% identity with Xenopus HSP30C. Northern blot analysis revealed that Xenopus HSP27 gene expression was developmentally regulated. Constitutive and heat shock-induced hsp27 mRNA accumulation was first detectable at the early tailbud stage while HSP27 protein was detected at the tadpole stage. Furthermore, hsp27 mRNA was enriched in selected tissues under both control and heat shock conditions. Whole mount in situ hybridization analysis detected the presence of this message in the lens vesicle, heart, head, somites, and tail region. Purified recombinant HSP27 protein displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of target proteins including citrate synthase, malate dehydrogenase and luciferase. Thus, Xenopus HSP27, like HSP30, is a developmentally-regulated heat-inducible molecular chaperone.     

Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in mouse alpha-crystallin gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of differential gene expression and mRNA stability in the developmental regulation of the hsp 30 gene family in heat-shocked Xenopus laevis embryos

Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33^oC for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tail-bud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37^oC for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximidetreated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development. © 1995 Wiley-Liss, Inc.     

Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.     

Nucleotide sequence of the engXCA gene encoding the major endoglucanase of Xanthomonas campestris pv. campestris

The nucleotide sequence of the gene (engXCA) encoding the major extracellular endoglucanase (ENGXCA) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X. c. campestris) was determined and compared with the N-terminal amino acid (aa) sequence of the purified enzyme. An open reading frame of 1479 bp encoding 493 aa was identified, of which the N-terminal 25 aa represent a potential signal peptide. Determination of the exact position of a Tn5 insertion within engXCA, which did not reduce the encoded enzyme activity, indicated that the C-terminal region of the protein is not crucial for ENGXCA activity. Comparison of the complete deduced aa sequence with those deduced from other endoglucanase- and exoglucanase-encoding genes revealed a region with a high degree of homology, located towards the C terminus of the protein. These data indicate that the X. c. campestris ENGXCA may have a domain structure similar to that of many other bacterial and fungal cellulolytic enzymes. Hydrophobic cluster analysis was performed on the deduced aa sequence. Comparison of this analysis with those of 30 other cellulase sequences belonging to six different families indicated that the X. c. campestris enzyme can be classified in family A. The two aa residues which had previously been identified as 'potentially catalytic' within this family of cellulases, are conserved in the X. c. campestris ENGXCA.     

Locus encoding a family of small heat shock genes in Caenorhabditis elegans: two genes duplicated to form a 3.8-kilobase inverted repeat.

The genes coding for hsp 16-48, previously identified by cDNA cloning, and for another 16-kilodalton heat shock protein designated hsp16-1 were characterized by DNA sequencing. The two genes were arranged in a head-to-head orientation. Both the coding and flanking regions were located within a 1.9-kilobase module which was duplicated exactly to form a 3.8-kilobase inverted repeat structure. The inverted repeat structure ended in an unusual guanine-plus-cytosine-rich sequence 24 nucleotides in length. The identity of the two modules at the nucleotide sequence level implies that the duplication event may have occurred recently. Alternatively, gene conversion between the two modules could also maintain homology of the two gene pairs. The small heat shock genes of Caenorhabditis elegans contained TATA boxes and heat-inducible promoters, the latter agreeing closely with the Drosophila melanogaster consensus sequence described by Pelham (Cell 30:517-528, 1982). Unlike the homologous D. melanogaster genes, each of these C. elegans genes contained a short intron, the position of which has been conserved in a related murine alpha-crystallin gene. The intron separated variable and conserved regions within the amino acid sequences of the encoded heat shock polypeptides.     

Xenopus hsp 70 genes are constitutively expressed in injected oocytes.

Xenopus heat-shock genes are transiently heat-inducible in somatic cells, but they are also subject to a long-term developmental control in oogenesis and early embryogenesis. In order to understand whether different genes or different promoter elements are involved in the two types of control, several genomic clones coding for Xenopus heat-shock proteins, hsp 70 and hsp 30, were isolated, characterised and tested for expression in oocytes and COS cells. Three isolated hsp 70 genes are nearly identical in their promoter and mRNA leader sequences, indicating that there is only one type of hsp 70 gene. These promoters contain a consensus sequence element (CT-GAA--TTC-AG) upstream of the TATA-box, which is presumably required for their transient heat-inducibility. The two isolated hsp 30 genes show 5'-flanking sequences similar to each other, except that one of them shows a homology disruption precisely around the consensus sequence element. The same gene contains a frameshift mutation in the protein coding part and, since it cannot be expressed after introduction into oocytes or COS cells, it is probably a pseudogene. The other hsp 30 gene is strongly heat-inducible in injected oocytes or transfected COS cells. In contrast, the hsp 70 genes are strongly heat-inducible in COS cells, but their expression is highly efficient in injected oocytes at the normal temperature and is not increased during heat shock. This represents correct cell type-specific regulation of a cloned reintroduced gene, since the endogenous hsp 70 genes are constitutively activated during oogenesis, leading to the accumulation of stored hsp 70 mRNA in oocytes.     

The HSP70 multigene family of Caenorhabditis elegans

1. The heat shock response of the nematode Caenorhabditis elegans has been characterized. 2. There are at least nine genes in the hsp70 multigene family of C. elegans. 3. Five of the hsp70 genes have been characterized and assigned to one of at least three hsp70 gene subfamilies. One of the subfamilies consists of an hsp70 protein that has the potential to be translocated into the endoplasmic reticulum and another subfamily consists of a protein that has the potential to be translocated into the mitochondria. 4. The C. elegans hsp70 multigene family has several unique characteristics including introns in the heat inducible hsp70 genes, at least one trans-spliced hsp70 mRNA and two grp78 related genes, one of which is highly heat inducible. 5. The identification and characterization of C. elegans hsp70 multigene family is the basis for a genetic characterization of the regulation and function of a gene family during the development of a multicellular eukaryote.     

Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K. Spacings and orientation of the coding regions as well as their gene expression in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts. Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.     

Cloning and characterization of two processed pseudogenes and the cDNA for the murine U1 snRNP-specific protein C

Genes for the snRNP proteins U1-70K, U1-A, Sm-B′/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3′ poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.     

Structure and expression of a human gene coding for a 71 kd heat shock ''cognate'' protein.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.