Organ-specific and ploidy-dependent somaclonal variation; a new tool in breeding

The organ-specific somaclonal variation means the differences between the variability of somaclones originated from different somatic tissue of plant. Significant differences in some agronomical characters were achieved among somaclones of seed and plumule meristem origin. The ploidy-dependent somaclonal variation means the differences between the variability of somaclones originated from different ploidy-level tissue. Increased variation among regenerated plants was postulated by origin from cultured cells of reduced ploidy level. The comparison of somaclonal variation in the progenies of diploid plants regenerated from callus of haploid and diploid origin supported the ploidy dependent theory. The pollenhaploid somaclone method (PHS-method) was developed and tested for utilization somaclonal variation in rice breeding. The PHS-method comprises the two well-known and widely applied in vitro methods which are the androgenesis (another culture) and genetic instability of cultured haploid somatic cells (callus cultures). Developmental varieties produced by this breeding sheme are under certification in Hungary.     

Identification of positive and negative regulatory regions controlling expression of the cartilage matrix protein gene.

A complex pattern of regulation of the cartilage matrix protein gene was revealed by transient expression experiments. A minimal promoter from positions -15 to +64 functioned in chondrocytes and fibroblasts. An enhancer located in the first intron exerted chondrocyte-specific stimulation on the minimal promoter activity. The same fragment, however, had a negative effect in fibroblasts. Between -334 and -15, a silencer was found which inhibited the gene expression driven from its homologous as well as heterologous promoters both in chondrocytes and fibroblasts. Additional positive and negative control regions were mapped further upstream of the promoter.     

Cloning and characterization of the HpaII methylase gene.

The HpaII restriction-modification system from Haemophilus parainfluenzae recognizes the DNA sequence CCGG. The gene for the HpaII methylase has been cloned into E. coli and its nucleotide sequence has been determined. The DNA of the clones is fully protected against cleavage by the HpaII restriction enzyme in vitro, indicating that the methylase gene is active in E. coli. The clones were isolated in an McrA-strain of E. coli; attempts to isolate them in an McrA+ strain were unsuccessful. The clones do not express detectable HpaII restriction endonuclease activity, suggesting that either the endonuclease gene is not expressed well in E. coli, or that it is not present in its entirety in any of the clones that we have isolated. The derived amino acid sequence of the HpaII methylase shows overall similarity to other cytosine methylases. It bears a particularly close resemblance to the sequences of the HhaI, BsuFI and MspI methylases. When compared with three other methylases that recognize CCGG, the variable region of the HpaII methylase, which is believed to be responsible for sequence specific recognition, shows some similarity to the corresponding regions of the BsuFI and MspI methylases, but is rather dissimilar to that of the SPR methylase.     

Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions.

U-snRNA genes in higher plants contain two essential promoter elements, the USE with sequence RTCCCACATCG and the TATA-like box, positioned in the -70 and -30 regions, respectively. Using an oligodeoxynucleotide containing the USE motif and oligodeoxynucleotides specific for the intragenic regions conserved in U-snRNAs, several sequences encoding U6 and U3 snRNAs were determined by polymerase chain reaction (PCR) amplification of Arabidopsis thaliana and tobacco genomic DNAs. This method provides a simple and rapid procedure for characterisation of plant U-snRNA genes and their promoters. It could also be used for the characterisation of other genes containing conserved upstream promoter elements. PCR-derived fragments were used as probes for the isolation of the U3 snRNA genes from a genomic library of Arabidopsis. Two isolated U3 genes were shown to be active when transfected into protoplasts of Nicotiana plumbaginifolia. Both U3 genes contain the USE and TATA-like upstream elements located in similar positions to the U6 genes of Arabidopsis. The encoded Arabidopsis U3 snRNAs can be folded into a secondary structure which is more similar to that of U3 RNAs from lower eukaryotes rather than from metazoa.     

7-2/MRP RNAs in plant and mammalian cells: association with higher order structures in the nucleolus.

Mammalian MRP (for mitochondrial RNA processing) RNA, also known as 7-2 RNA, is a nuclear encoded small RNA which has been reported to function in two different cellular compartments: in the mitochondria and in the nucleus. The ribonucleoprotein particle which contains the 7-2/MRP RNA, called RNase MRP, has ribonucleolytic activity and shares some structural similarity with RNase P. It has been proposed that in mitochondria, the RNase MRP is responsible for endonucleolytic cleavage of primer RNA during DNA replication. We have characterized the gene and cDNAs encoding 7-2/MRP-like RNA in Arabidopsis and tobacco, and found that in plants this RNA is enriched in nucleoli but is undetectable in purified mitochondria isolated from tobacco leaves or cells grown in suspension. In glycerol gradients tobacco 7-2/MRP RNA cosediments with large approximately 80S structures possibly representing ribosomal precursors. Fractionation of HeLa cells has also revealed that 7-2/MRP resides in the nucleolus and that most of it is associated with complexes sedimenting at approximately 80S, similar to those containing the U3 nucleolar RNA which is known to participate in pre-rRNA processing. These results indicate that the 7-2/MRP ribonucleoparticle may be involved in ribosome biogenesis, in both plant and mammalian cells.     

Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l(1)2Bc, and l(1)2Bd) alleles, and classes of noncomplementing (npr 1) and partially noncomplementing l(1)2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the l(1)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.     

Cellular localization of bovine pancreatic trypsin inhibitor and related molecular forms in bovine lung

Summary In addition to bovine pancreatic trypsin inhibitor (BPTI), three BPTI-related molecular forms (isoinhibitors I, II and III) were isolated from bovine lung by affinity chromatography on immobilized trypsin and subsequently purified by Fast Protein Liquid Chromatography. These inhibitors are identical to the isoinhibitors previously isolated from bovine spleen. Their localization in bovine lung was studied by immunohistochemical techniques, using two different immunoglobulin preparations, selectively recognizing BPTI or the other molecular forms.BPTI-related immunoreactivity was found to be restricted to isolated cells, often identified as mast cells by Toluidine Blue staining. In contrast, isoinhibitor-related immunoreactivity, which also occurs in the mast cells, is present in a number of other cell types. These types include: (i) the smooth muscle cells of different calibre vessels, (ii) the ciliated cells of the bronchial epithelium and the related mucus, and (iii) many cells at alveolar level.Comparison of these data with previous results obtained for bovine spleen suggest multiple physiological roles for these inhibitors.