The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.     

The porcine proteasome subunit A4 (PSMA4) gene: isolation of a partial cDNA, linkage and physical mapping

A partial cDNA clone encoding the porcine proteasome subunit A4 ( PSMA4 or proteasome subunit C9) has been isolated from a porcine muscle cDNA library and sequenced. A biallelic Taq I RFLP was identified in Large White, Landrace and Duroc breeds. Moreover, the 3'-untranslated region of the gene showed a triallelic SSCP. By linkage analysis the PSMA4 locus was assigned to pig chromosome 7 and by radioactive in situ hybridization this locus was mapped to the region 7q13–q14.     

Cloning of heat-shock locus 93D from Drosophila melanogaster.

Using the microcloning approach a number of recombinant lambda phages carrying DNA from the 93D region have been isolated. Screening genomic libraries, cloned in phage lambda or cosmid vectors, with this isolated DNA yielded a series of overlapping DNA fragments from the region 93D6-7 as shown by in situ hybridization to polytene chromosomes. In vitro 32P-labelled nuclear RNA prepared from heat-shocked third instar larvae hybridized specifically to one fragment within 85 kb of cloned DNA. The region which is specifically transcribed after heat shock could be defined to a cluster of internally-repetitive DNA and its neighbouring proximal sequences. Over a sequence of 10-12 kb in length the DNA is cut into repeat units of approximately 280 nucleotides by the restriction endonuclease TaqI. The TaqI repeat sequences are unique in the Drosophila genome.     

Ribosomal DNA spacer-length variation inSecale spp. (Poaceae)

Variation in ribosomal DNA spacer length was analysed in 23 populations of 12Secale spp. by restriction enzyme analysis. Digestion of rDNA with Taq I endonuclease enzyme yields spacer fragments that include the subrepeat array and the non-repetitive region downstream of the array. Extensive spacer length variation existed in most species with Taq I fragment lengths ranging from 0.9–3.1 kb. These length variants have been attributed to the differences in number of 134 bp spacer subrepeats within rDNA arrays.S. silvestre was the only species to exhibit a unique spacer length variant of 0.9 kb and this was shown to result from the presence of an extra Taq I site in the spacer. rDNA spacer length frequencies were determined for the species. These frequencies were used to derive phenetic relationships between the species by numerical taxonomic methods. In plots constructed fromGower's distance matrices,S. silvestre appeared well separated from the major cluster consisting of the other species. On the basis of morphological and cytogenetic criteria,S. silvestre is considered the most ancient species. The rDNA data is consistent with this interpretation as it shows a clear differentiation ofS. silvestre from all the other species based on length and nucleotide sequence composition of the spacer region.     

Sequences homologous to the human D1S1 locus present on human chromosome 3.

We have investigated the segregation, in somatic cell hybrids, of the human D1S1 locus, previously assigned to 1p36 by in situ hybridization. We have shown that the clone which defines this locus, lambda Ch4A-H3, originates from human chromosome 3, but contains a 1.7-kilobase (kb) PstI-HindIII repetitive element that is also present on chromosome 1, probably distal to PGD. The clone recognizes restriction fragment length polymorphisms within the single-copy sequence on chromosome 3 and one for the enzyme StuI in the repeated sequence on chromosome 1. These experiments thus expose a level of complexity in the D1S1 locus not revealed by earlier in situ hybridization studies.     

Identification of an H-2Kd gene using a specific cDNA probe.

A cDNA clone known to code for a mouse histocompatibility (class I) antigen was found to contain a sequence specific for a subpopulation of H-2 genes. This unique sequence is located in the 3'' non-coding region close to the stretch of poly(A) nucleotides. A subclone containing this fragment (pH-2d-5) has been used to select hybridizing mRNA. Translation of the mRNA in vitro shows that H-2Kd mRNA is selected. Southern blot analysis of DNA from congenic recombinant mice show that at least one gene containing this sequence is located at the K locus (region) of the major histocompatibility complex. This gene contains a 3.7-kb BglII and a 13-kb EcoRI restriction endonuclease fragment. This gene has been isolated from a genomic DNA library.     

Restriction fragment length polymorphism at the HLA-C locus

We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde 1, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLLs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu lI site in the fifth intron.