Cellular localization of the embryo-specific hybrid PRP from Zea mays, and characterization of promoter regulatory elements of its gene.

The expression, regulation and cellular localization of ZmHyPRP, a gene marker of embryo differentiation whose expression declines after ABA induction, was studied. ZmHyPRP is a proline-rich protein with a C-terminal domain having eight cysteines in a CM8 pattern. Transient expression in onion epidermal cells, transformed with a 2x35S::ZmHyPRP-GFP construction, indicated the protein is present in vesicles lining the membrane of the cell. The ZmHyPRP gene expression is under the control of classic promoter seed-specific regulatory elements such as Sph/RY and G-boxes, suggesting regulation by B3 and b-ZIP transcription factors. Promoter deletion analysis, by particle-bombardment transient transformation of maize immature embryos with serial deletions of the promoter fused to GUS, showed the presence of two negative regulatory elements, NE1 (-2070 to -1280) and NE2 (-232 to -178), in the ZmHyPRP promoter. By selective deletion or mutation of ZmHyPRP regulatory promoter elements we conclude that the promoter expression is attenuated by the NE2 element as well as by the G-box2 and the Sph1-2 box together with the G-box2.

Effect of histone composition on the stability of chromatin structure.

The mode of fragmentation of chromatin by micrococcal nuclease has been studied in nuclei from different sources at physiological ionic strength and low temperature. During digestion, the size of chromatin was reduced until an average S value of 95-100 (hen erythrocyte) or 60-65 (rat liver) was attained. The accumulation of these structures correlated with the period of maximum solubility (80%), indicating that the bulk of chromatin behaved in this manner. Further digestion did not result in a corresponding decrease in S value but in a bimodal sedimentation pattern. As opposed to this behavior, chromatin containing actively acetylated core histones showed a continuous variation in size during the digestion. Indirect immunoprecipitation of chromatin by anti-H5 antibody and sheep anti-rabbit antibody revealed that the acetylated chromatin is partially depleted of H5.

The chromatin of sea urchin sperm.

Digestion of sea urchin sperm nuclei with micrococcal nuclease yields nucleosomal monomer fragments of 151 and 164 base pairs. Prior trypsin treatment of the sperm chromatin does not alter the size of these monomer DNA fragments despite the fact that the H1 histone is reduced to a limit globular peptide of about 83 residues. Heterologous reconstitution experiments show that this peptide is capable of protecting an extra 22 base pairs beyond the core particle in a chromatosome. Nuclease digestion of reconstitutes from DNA and sperm core histones yields a core monomer of about 141 base pairs. It is concluded that this sperm chromatin contains a chromatosome of 164 bp essentially similar to that observed in the more usual chromatins. Edman degradation of the H1 limit peptide shows its sequence to be closely analogous to the corresponding peptide of calf H1 and chicken H5. Circular dichroism studies of histone H1 from the sperm of three sea urchin species demonstrate the presence of trypsin-sensitive helical regions outside the globular domain that are absent in calf H1 and chicken H5.

The interaction of histone H3 with histone H4 and with other histones studied by 19F nuclear magnetic resonance.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3-H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and h4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3-H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1-0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.

Developmental and Hormonal Regulation of Genes Coding for Proline-Rich Proteins in Female Inflorescences and Kernels of Maize

The pattern of expression of two genes coding for proteins rich in proline, HyPRP (hybrid proline-rich protein) and HRGP (hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRP mRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments.

Adsorption of recombinant human bone morphogenetic protein rhBMP-2m onto hydroxyapatite.

Recombinant human mature bone morphogenetic protein 2 (rhBMP-2m) has been expressed to study its adsorption onto precipitated hydroxyapatite (HA). The influence on the adsorption process of different parameters such as pH and concentrations of calcium, phosphate or NaCl has been investigated. Although the adsorption proceeds rapidly at the initial stages, the maximum adsorbed amount is reached after four hours. The process is notably influenced by adding calcium or phosphate to the system but, while calcium ions increase the adsorption of rhBMP-2m onto HA, phosphate ions inhibit it. The influence of pH and NaCl concentration are notable but less important than those of calcium and phosphate. The adsorption data fit well to a Langmuir adsorption isotherm. The values of the isotherm parameters have been calculated and discussed, and an adsorption mechanism has been proposed.

Characterization of antigenic polypeptides of the RNP, Sm and SS-B nuclear antigens from calf thymus.

Antinuclear autoantibodies are a hallmark of autoimmune diseases. The RNP, Sm and SS-B nuclear antigens from calf thymus in whole tissue, nuclear extracts and fractions have been studied by using different techniques including immunodiffusion, counterimmunoelectrophoresis and protein blotting. Such studies were done in order to obtain a precise characterization of the polypeptide components of those antigens. From our results it can be established that: one 69.8 Kd polypeptide (for whole tissue and nuclei) and a number of well-defined 32-38-Kd polypeptides (for nuclear extracts and ammonium sulfate fractions) show an antigenic character against anti-RNP sera; anti-Sm sera from different patients show in all cases a variable component of antigenic polypeptides, including one 28.8, 29.7 Kd doublet and two singlets of 14.8 and 11.0 Kd; and a 52.0-Kd SS-B antigenic polypeptide is found for whole tissue, which is gradually degraded in nuclei and nuclear extracts to a more stable 47.1-Kd polypeptide.

Molecular analysis of a putative transposable retroelement from the Zea genus with internal clusters of tandem repeats.

The molecular characterization of a recently discovered family of long repetitive sequences, termed ZLRS, is described. These elements belong to the class of moderate dispersed repetitive DNA and are specific to the Zea genus. An 8089-bp sequence from a Zea diploperennis ZLRS element have been elucidated. Sequence analysis reveals the presence of a long terminal repeat-like region, two clusters of different tandem repeats and several ORFs. On these grounds, ZLRS could be considered a new member of the superfamily of transposable retroelements. Tandems are present in the majority of ZLRS elements, they show an important stem-loop secondary structure predicted by the computer and their sequence conservation suggests a functional role.

The Tub alpha 3 gene from Zea mays: structure and expression in dividing plant tissues.

A gene (Tub alpha 3) coding for an alpha-Tub, expressed in dividing tissues, has been cloned from Zea mays. The deduced amino acid (aa) sequence, 450 aa long, is very similar to the other plant alpha-Tub (85-89% homology) so far reported, and in particular to the other two aa sequences (alpha 1-Tub and alpha 2-Tub) already published from the same species (93% homology). The genomic structure is also very similar, having three introns located at the same positions as in the Tub alpha 1 and Tub alpha 2 genes, one of them placed at the same position in the homologous genes from Arabidopsis thaliana. Nevertheless, the noncoding sequences are very different from the two other maize genomic sequences. In particular, no homology has been found either in the 5' upstream or in the 3'-untranslated sequences. Using specific 3' probes, it has been possible to detect the mRNA coded by this gene in many of the plant organs measured, but its highest abundance is observed in the organs rich in dividing cells, a pattern correlated with that of the histone H4-encoding gene. A cDNA clone has been identified in maize coleoptiles and sequenced, confirming the expression of the Tub alpha 3 in this organ. No preferential accumulation in any organ of the plant was found, in contrast with what was observed in the Tub alpha 1 and Tub alpha 2 genes already described. The Tub alpha gene family seems to consist in maize by at least two groups of homologous sequences, each one including a maximum of two or three coding units.

Structure, organization and expression of the eukaryotic translation initiation factor 5, eIF-5, gene in Zea mays.

The maize genomic DNA sequence encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from genomic library of maize seedlings and the exon-intron structure determined (accession number AJ132240). The length of genomic DNA sequenced was about 7kb and contained two exons with the translation start site in exon 2. The only intron is located in the non-coding 5' region and it is 1298bp long with the splice acceptor and donor sites conforming to the AG/GT rules. Repetitive sequence fragments are located in the 5' and 3' intergenic region. The accumulation of eIF-5 mRNA was studied by RNA blot and in situ hybridization. The observed distribution of mRNA may correlate with the function of the protein, as it appears to be highly abundant in tissues where the proportion of cells actively dividing is very high, such as meristematic regions.

Characterization of the sequence coding for the clathrin coat assembly protein AP17 (sigma2) associated with the plasma membrane from Zea mays and constitutive expression of its gene.

The cDNA and genomic sequences coding for the clathrin coat assembly protein AP17 (sigma2) from maize and its corresponding mRNA accumulation have been analyzed. This protein in yeast and mammals has been shown to be part of the associated protein (AP) complex of clathrin in the plasma membrane. The availability of this sequence as well as a previous AP19 in a plant allows one to propose that specific AP complexes exist in plants in the Golgi complex and in the plasma membrane. The AP17 protein is encoded in maize by a single gene, and its mRNA accumulates in all the organs studied. In the immature embryo, it displays a pattern of expression typical of constitutively expressed genes.

Multiple variability in the sequence of a family of maize endosperm proteins.

A collection of cDNA clones, corresponding to a group of maize endosperm proteins classified in the glutelin-2 (or reduced soluble proteins) and in the zein-2 subfractions, has been identified and characterized. The nucleotide sequence of three of these clones has been obtained and the amino acid sequence deduced. They appear to correspond to a small family of genes that are specifically expressed in immature endosperm simultaneously to zeins, the best characterized proteins from this tissue. Unlike zeins, the proteins of the glutelin-2 and zein-2 family contain sequences homologous to storage proteins from other cereals such as gliadins or hordeins. The cDNA clones encoding for the two types of proteins have been compared, and a high degree of homology has been observed for both the nucleotide and amino acid sequences. The differences existing in both the coding and non-coding regions allow the definition of multiple types of variability in their sequence. An hypothesis is proposed on how sequence diversity may have been generated in this particular class of plant proteins.

A new family of repetitive nucleotide sequences is restricted to the genus Zea.

We have isolated a new family of moderately repetitive nucleotide sequences (about 2500 copies per haploid genome) specific to the genus Zea and absent in other graminaceous species. These sequences are interspersed in the genome and they show the same genomic organization pattern and similar copy number in all the Zea species examined. These two facts, consistency in the copy number and the same organization pattern, would indicate on the one hand that these sequences were amplified before the divergence of Zea species, and on the other hand that maize and all the teosintes could be considered as the same evolutionary population. Independent clones corresponding to the repetitive sequences have been isolated and sequenced from a genomic library of the teosinte, Zea diploperennis. The repeats, flanked by HaeIII sites, are more than 70% G + C-rich, on average 253 bp long and show 78% similarity to each other. These repetitive sequences are in a highly methylated-C context and they present some features resembling those of coding sequences, such as high CpG and low TpA content, and similar codon usage to maize genes in one of the reading frames. Moreover, the repetitive probe hybridizes with RNA extracted from different tissues of maize and from teosinte, indicating that these repeats or similar ones are present in transcribed sequences.

Structure and characterization of the gene encoding the ferredoxin-NADP reductase-binding protein from Zea mays L.

A genomic clone encoding ferredoxin-NADP reductase binding protein (BP) from Zea mays L. was sequenced and characterized. The promoter region (692 bp) shows several motives resembling those involved in enhancement, tissue-specific expression and light regulation in plants, besides the typical TATA and CAAT boxes. The coding sequence is interrupted by two introns. The deduced amino acid (aa) sequence corresponds to 22.85 kDa for the precursor polypeptide, including a transit peptide of 64 aa and a mature protein of 148 aa.

A pair of genes coding for lipid-transfer proteins in Sorghum vulgare.

Approximately five genes coding for lipid-transfer proteins (LTP) can be detected in Sorghum vulgare by DNA blots using a specific genomic probe. Two of these genes have been identified and sequenced. The two genes (ltp1 and ltp2) code for very similar (91.8% identity) proteins, they are separated by approx. 4 kb of DNA and their open reading frames may be read in the same direction. The gene (ltp1) located upstream has an intron placed in the same position already described for other ltp in maize and rice. Gene ltp2 has no intron. cDNAs corresponding to ltp1 have been identified in a 6-day-old plantlet library, but not for ltp2. The results of the comparison between the two sequences indicate the presence of a gap between the two genes in their promoter region. LTP seem to be coded for in plants by a small family of genes. At least in sorghum, two of its components are tightly clustered in the same genomic region.

Characterization of a rice gene coding for a lipid transfer protein.

The cloning and sequence analysis of a gene that encodes a lipid transfer protein (LTP) from rice is reported. A genomic DNA library from Oryza sativa was screened using a cDNA encoding a maize LTP. One genomic clone containing the gene (Ltp) was partially sequenced and analyzed. The open reading frame is interrupted by an 89-bp intron. From the results of Southern hybridizations, Ltp appears to be a member of a small multigenic family. Transcripts of the corresponding gene were detected in several tissues including coleoptile, leaf, endosperm, scutellum and root. The transcription start point was determined by primer extension. The deduced amino-acid sequence of the Ltp product is shown to be homologous to LTPs from other crops.

Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing.

We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.

Multiple polyadenylation sites are active in the alpha 1-tubulin gene from Zea mays.

Two cDNA clones (MR2 and MR29) encoding the same alpha-tubulin isotype (alpha 1) have been identified and characterized from maize roots. The sequence of these two cDNA clones is identical to a previously described cDNA clone (MR19) except in the location of their polyadenylation site. The sequence of the cDNA and its homologous genomic clone (MG19/14) shows two putative polyadenylation signals which could direct the variable 3' processing of the observed transcripts. Endonuclease S1 protection analysis in this 3' flanking region confirms the presence in the alpha 1-tubulin gene from Zea mays of these two main functional polyadenylation sites and possibly other related ones. The relative accumulation of RNAs bearing the two main polyadenylation sites has been tested by using a RNA-slot analysis of several tissues of the plant. It appears that a higher proportion of shorter mRNA species is found in actively dividing tissues.

Rapid changes induced in developmental programmes of the maize embryo detected by analysis of the expression of genes encoding proline-rich proteins.

The pattern of expression of two genes coding for proline-rich proteins, zmHyPRP and zmHRGP, in Zea mays is modified when the embryogenesis programme is altered by placing the embryos in conditions which promote either precocious germination or callogenesis. zmHyPRP gene expression is rapidly arrested when the embryogenesis programme is altered. zmHRGP mRNA is highly induced in scutellum within a few hours of callogenesis or precocious germination.

Isolation of a 167 basepair chromatosome containing a partially digested histone H5.

A test has been made of the proposal that protection of the 167 basepair DNA length in the 'chromatosome' is due only to the central globular domain of the lysine-rich histones. Chicken erythrocyte chromatin was treated with trypsin to leave only the limit peptide from histones H1 and H5. Nucleosome monomers were then isolated on sucrose gradients following micrococcal nuclease digestion and were found to contain the 167 basepair DNA band as in intact chromatin. The presence of the limit peptide from H5 on the monomers was confirmed using an antibody to H5. It is concluded that the trypsin-susceptible domains of the lysine-rich histones are not involved in the protection of the 2-turn 167 basepair length of DNA in the nucleosome.