Drosophila 5 S RNA processing requires the 1-118 base pair and additional sequence proximal to the processing site.

Using an in vitro processing system, we have identified a required sequence surrounding the Drosophila melanogaster 5 S RNA processing site at nucleotide 120. Mutations in this region vary the processing rate from complete inhibition to a level equal to or greater than wild type. Analysis of mutants at +1 and in the region 118-122 separates the inhibitory effect into two parts. 1) Nucleotide 118 C, the base-paired nucleotide in helix I proximal to the processing site, plays an essential role. Changing it to a purine inhibits processing. The +1-118 base pair must be intact, but this alone is not sufficient for processing, since compensatory changes at +1 do not restore down-processing mutants at 118 to the wild type level. 2) The processing site has to be pyrimidine rich; multiple contiguous purines inhibit processing. On the other hand, multiple pyrimidines can largely negate the inhibitory effect of a mutation at position 118. Thus a base-paired C at 118 followed by a stretch of pyrimidines is the processing signal, which may be recognized by the processing enzyme and/or a required accessory factor.

In vitro processing of Drosophila melanogaster 5 S ribosomal RNA. 3' end effects and requirement for internal domains of mature 5 S RNA.

5 S RNA processing in Drosophila melanogaster, the removal of 15 nucleotides from the 3' end of the 135-nucleotide (nt) primary transcript, may play an important role in the regulation of 5 S RNA transport and ribosome assembly. We have uncoupled processing from transcription using gel purified primary transcripts processed in vitro by a cellular S100 extract. The RNA was generated by a homologous transcription system or by a T7 RNA polymerase reaction using a constructed 5 S RNA gene linked to a T7 promoter. In vitro D. melanogaster 5 S RNA processing is heat- and EDTA-sensitive, suggesting a requirement for protein, and produces a 3' end characteristic of mature 5 S RNA. Processing of substrate RNAs with altered 3' ends shows that the 3'-U5 tail (nt 131-135) inhibits the reaction. 30 nt, including all of domain IV and most of domain V, are dispensible for processing, whereas deletions including the base of stem V and all or part of stem III severely inhibit it. Several possible mechanisms are discussed.

Visualization of RNA binding proteins by sequential gel shift and ultraviolet cross-linking.

RNA binding proteins partially constitute the ribonucleoprotein or protein machinery for RNA processing (splicing, polyadenylation and 3' end formation), transport, and storage. We have devised a novel method for the detection of RNA binding proteins in vitro. The template for transcription is a cloned Drosophila melanogaster 5S rRNA gene. The method is a two-dimensional gel analysis involving: in vitro transcription of 32P-labeled 5S rRNA using a cellular S-100; resolution of labeled RNA protein complexes from unbound RNA on a first-dimension mobility shift gel; cross-linking of RNA to protein in gel by ultraviolet irradiation; degradation of the RNA by RNase A and T1; and analysis of 32P-protein patterns on a second-dimension discontinuous SDS gel by autoradiography. The pattern of proteins associated with 32P-5S rRNA is obtained by covalent transfer of 32P-nucleotides from RNA to the proteins with which the RNA was bound. This method could be useful in the analysis of RNA maturation and processing pathways.

Human protein binding to DNA sequences surrounding the human T-cell lymphotropic virus type-I long terminal repeat polyadenylation site.

The long terminal repeats (LTRs) of RNA tumor viruses, including human T-cell lymphotropic virus type I (HTLV-I), contain the control elements for expression of viral genes. Sequence-specific LTR-DNA-binding proteins could regulate viral functions. To search for such proteins we have used an in vitro non-denaturing polyacrylamide gel assay, with restriction fragments of the HTLV-I LTR and nuclear protein extracts from several HTLV-I-infected cell lines and an uninfected T-cell line, H9. Four DNA-binding activities were observed, including non-specific DNA-binding activity and at least two activities (forms I and II) which bind specifically to a HinfI restriction fragment from nucleotides +181 to +334 relative to the transcription start site. DNA-binding activities I and II were partially resolved by ion-exchange chromatography and mapped by protection experiments to two 10-20-bp blocks surrounding the polyadenylation site at +221. Of the cell lines tested, form II was abundantly found in C10/MJ, and forms I and IV were also found in C91/PL, C81-66-45, MT2 and H9 cells.

A unique nucleoprotein structure associated with the Drosophila melanogaster 18-28 S rDNA nontranscribed spacer.

We have detected unique nucleoprotein particles specific for the 18-28 S rDNA nontranscribed spacer of Drosophila melanogaster. The particles migrate between di- and trinucleosomes on nucleoprotein gels, and are between mono- and dinucleosomal in DNA length. These migration properties suggest that the nontranscribed spacer particles could have a protein component larger than a histone core. The variant nucleoprotein structures map primarily within the nontranscribed spacer 235 base pair internal subrepeat, which is AT-rich and possesses a 50 base pair sequence homologous to the RNA polymerase I binding site.

D1 protein of Drosophila melanogaster. Purification and AT-DNA binding properties.

D1 protein of Drosophila melanogaster is a sequence-specific DNA-binding protein which recognizes AT-rich DNA sequences. AT-rich DNA sequences in eukaryotic organisms are distributed in two characteristic ways: flanking transcriptional units and in constitutive heterochromatin. D1 could play a role in regulation of gene expression and in geographical localization of DNA sequences within the nucleus. D1 has been partially purified by ion exchange chromatography. DNA-binding activity was investigated by nucleoprotein gel electrophoresis, using end-labeled restriction fragments varying in AT sequence content. D1 binds most tightly to the satellite sequence -AATAT-, with intermediate strength to the complex satellite (359-base pair repeat) and another AT-rich (68% AT) mixed sequence DNA, and least to the simple satellite sequence -AAGAG-.

Superstructural differences between chromatin in nuclei and in solution are revealed by kinetics of micrococcal nuclease digestion.

Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.

Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines.

Micrococcal nuclease digestion was used to analyze Epstein-Barr virus (EBV) DNA structure in nuclei of transformed cells. Digests of virus-producing (P3HR-1), non-virus-producing (Raji), and superinfected Rajii cell nuclei were fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to 32P-labeled EBV DNA. The viral DNA of Raji nuclei produced a series of bands on electrophoresis whose lengths were integral multiples of a unit size, which was the same as the repeat length of host DNA. Viral DNA in nuclei of P3HR-1 and superinfected Raji cells produced faintly visible bands superimposed on a smear of viral DNA which dominated the hybridization pattern. No differences were detected in the patterns when total DNA digests from Raji, P3HR-1, and an EBV DNA-negative cell line (U-698M) were analyzed by ethidium bromide staining or by hybridization with the use of 32P-labeled lymphoblastoid cell DNA as probe. We conclude that the EBV episomal DNA of Raji cells is folded into nucleosomes, whereas most of the viral DNA of P3HR-1 and superinfected Raji cells is not. This pattern of DNA organization differs signficantly from that in papova group viruses.

Cross-referencing testis-specific nuclear proteins by two-dimensional gel electrophoresis.

Discontinuous sodium sodecyl sulfate-gel electrophoresis combined with acid-urea gel electrophoresis reveals that both testis histone H1 classes TH1-X (Branson, R. E., Grimes, S. R., Jr., Yonuschot, G., and Irvin, J. L (1975) Arch. Biochem. Biophys. 168, 403-412) and H1 contain two polypeptides each. Migration properties and relative staining intensities of the four H1 histone proteins in testis support the following conclusions. 1. Two testis-specific forms become the major H1 components at some stage of spermatogenesis. 2. During this stage they assume structural and functional role analogous to those of their two somatic counterparts. We have also detected a new testis-specific protein containing cysteine.