The yeast nucleolar protein-encoding gene NSR1 was isolated by low-stringency screening of a yeast genomic library with the human heterogeneous nuclear ribonucleoprotein type A1 (hnRNP A1) cDNA probe, and was mapped to chromosome VII. RNA abundance was determined and the transcription start point and polyadenylation site were mapped. A comparison between the Nsr1 and hnRNP A1 proteins, based on homopolymer RNA binding to their structural domains in vitro, revealed a striking biochemical similarity. When the N-terminal, lysine- and arginine-rich domain of Nsr1 was removed, the truncated protein behaved similarly to hnRNP A1; furthermore, the two RRM (RNA recognition motif) domains of Nsr1 behaved in the same manner as the two RRM domains of hnRNP A1. The biochemical data, therefore, would support the hypothesis that the two RRM domains in hnRNP A1 and Nsr1 interact with RNA in a similar manner in both mammalian and yeast cells, respectively.

A message that geneticists and molecular biologists are increasingly addressing to plant breeders is that the plant genome is remarkably unstable. Plants of a population within a species can show karyotypic variations, with changes in chromosome number or with the appearance of supernumerary or β-chromosomes; but extensive submicroscopic variations, such as movement of transposable elements, chromosome rearrangements and gene amplification may also occur (Durrant 1974; Price et al. 1983; Walbot and Cullis 1983; Marx 1984).

Heterogeneous nuclear ribonucleoprotein (hnRNP) core protein A1 is a major component of mammalian hnRNP 40 S particles. We describe the structure of an active A1 gene and report on the partial characterization of the A1 gene family. About 30 A1-specific sequences are present per haploid human genome: 15 such sequences were isolated from a human genomic DNA library. Many corresponded to pseudogenes of the processed type but by applying a selection for actively transcribed regions we isolated an active A1 gene. The gene spans a region of 4.6 x 10(3) base-pairs and it is split into ten exons that encode the 320 amino acid residues of the protein. The amino acid sequence derived from the exon sequences is identical with that deduced from cDNA and reported for the protein. One intron exactly separates the two structural domains that constitute the protein. Each of the two RNA-binding domains in protein A1 is encoded by one exon. Experimental evidence indicates that the A1 gene can encode for more than one protein by alternative splicing. The gene is preceded by a strong promoter that contains at least two CCAAT boxes and two possible Sp1 binding sites, but it lacks a TATA box.

In eukaryotic cells transcript processing is strictly dependent upon binding of specific proteins. Nuclear RNA binding proteins share a common domain, which is involved in RNA binding. In order to characterize RNP-RNA interactions we have performed a secondary structure prediction based both on statistical algorithms and comparative analysis of different proteins. A high conservation for secondary structure propensity between different RNPs was observed.

Protein A1 is one of the major component of mammalian ribonucleoprotein particles (hnRNP). Human protein A1 cDNA cloning and sequencing revealed the existence of at least two protein isoforms. Among the cDNAs examined, sequence differences were found both in the structural portion, leading to aminoacid changes (Tyr to Phe or Arg to Lys) and in the non translated 3'-region where two T-stretches of different length were observed. Interestingly one of the aminoacid substitutions falls into a consensus sequence common to many RNA binding proteins. Northern blot analysis of poly A+ RNAs from five human tissues revealed two mRNA forms of 1500 and 1900 n due to alternative polyadenylation. Analysis of genomic DNA showed at least 30 A1-specific sequences, some of which correspond to processed pseudogenes. These results suggest that protein A1 is encoded by a multigene family.

Highly repeated nuclear DNA sequences from suspension cultured cells of Oryza sativa L. cv. ‘Roncarolo’ have been cloned in pBR322. Ten clones with specific digestion patterns have been randomly selected. Nine sequences appear to be organized in a clustered tandem array while one is interpersed in the rice genome. The clones have been used to gather information on: (a) their modulation in cultured cells as compared to whole plant and (b) their distribution in different rice cultivars belonging to the Japonica or Indica subspecies of Oryza sativa L. Hybridization with nuclear DNA isolated either from suspension or from seedlings of the ‘Roncarolo’ cultivar revealed extensive quantitative variations, with most cloned sequences showing amplification (up to 75-fold) in cultured cells. Hybridization with nuclear DNA isolated from seedlings or suspension cultured cells from different cultivars belonging to the Japonica or to the Indica sub-species of O. sativa have shown that (a) amplification also occurs in a similar pattern in the case of DNA from the other tested suspension cultured cell types but not in the case of DNA from seedlings; (b) in some cases the tested sequences show minor but significant variations in different rice accessions.

As we have previously demonstrated, mammalian single stranded DNA binding proteins (ssDBP) and heterogeneous nuclear RNA binding proteins (hnRNP proteins) are antigenically and structurally related. In this paper we show that ssDBP are specific proteolytic products of hnRNP core proteins. Proteolysis can be observed in crude extract, both total and nuclear and is not inhibited by the most commonly used protease inhibitors. Such phenomenon can be observed in HeLa cells, human fibroblasts and calf thymus extracts. A trypsin-like protease that cleaves purified hnRNP proteins to give ssDBP of Mr = 24-28 Kd can be purified from HeLa cells. A precursor-product relationship can be established between hnRNP core proteins type A and an ssDBP of 24 Kd (UP1).

Antibodies induced against mammalian single‐stranded DNA binding protein (ssDBP) UP I were shown to be cross‐reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti‐ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re‐evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to study the fate of the parental genomes in somatic cell hybrids between distantly related species, protoplasts from cultured cells of Daucus carota and Oryza sativa were fused. Selective conditions resulted, exclusively, in the growth of hybrid colonies which combined the capacity to multiply of carrot cells with the natural resistance to A2CA of rice cells. A methodology for measuring the relative contribution of the parental cells to the hybrid nuclear genome has been worked out. This is based both on hybridization of nuclear DNA bound to nitrocellulose filters (dot hybridization) with radioactively labelled nuclear DNA from one of the parents and on agarose gel fractionation of nuclear DNA digested with restriction endonucleases. The dot hybridization analysis, performed on one of the D. carota x O. sativa cell hybrids, showed that the major portion of the nuclear genome is homologous with the carrot partner, with rice contributing only a minor fraction, along with the selected resistance gene(s). The homology was confirmed after agarose gel fractionation of restriction endonuclease BamHI-digested nuclear DNA. Furthermore, strong homology at the level of gene expression between hybrid and carrot cells was shown by polyacrilamide gel electrophoresis of total soluble proteins.

Antibodies were raised in rabbit against a pure subset of calf thymus single-stranded DNA binding proteins (ssDBPs) and purified by affinity chromatography on antigen-Sepharose. In Western blot experiments these antibodies were shown to react to the same extent with the whole family of bovine ssDBPs, as well as with ssDBPs from HeLa cells. When used to stain total cell extracts from both calf thymus and HeLa cells the antibodies reacted only with bands corresponding to the ssDBPs and with a set of bands of higher molecular weight, whose electrophoretic pattern matched that of the 40S hnRNP core proteins. In effect we observed that purified 40S hnRNP core proteins from HeLa cells were strongly reactive with the antibodies. Moreover after partial tryptic digestion HeLa cells ssDBPs and hnRNPs produced immunoreactive fragments of the same molecular weight and isoelectric point. Extensive structural homologies can thus be evidenced between these two classes of proteins, which share the property of selective binding to single-stranded nucleic acids.