Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

Affinity modification of Escherichia coli ribosomes by 4-[(N-2-chloroethyl)N-methylamino]benzyl-5'-phosphamide hexauridylate in a complex stabilized by codon-anticodon interactions on P and A sites

Affinity labeling of E. coli ribosomes with 4-[(N-2-chloroethyl)-N-methylamino] benzyl-5'-phosphamide of hexauridylate was studied within the complex containing tRNAPhe at P site and Phe-tRNAPhe at A site directed by EF-Tu and GTP. Ribosomal proteins as well as rRNA both in 30S and 50S subunits were found to be labelled within the complex. Labeled proteins were identified as S3, S9 and L2. Selectivity of affinity labeling with mRNA analogs was shown to depend on the functional state of the ribosomes. Modification was more selective within the complex stabilized by codon-anticodon interaction both at A and P-sites than within the complex in which this interaction takes place preferentially at P site.     

Myelotoxicity of epirubicin]

Epirubicin (pharmorubicin, India), an antitumor antibiotic of the anthracycline group, was studied in regard to its effect on peripheral blood, bone marrow and lymphoid organs (the thymus and spleen) of CBA mice after its intraperitoneal administration in a single dose equal to the MIC (7.8 mg/kg) and in a course dose (1/5 of the MIC 5 times a day). The cytogenetic impairments induced by the cytostatic were estimated on metaphase plates with the bone marrow specimens and by counting the peripheral blood erythrocytes with micronuclei (the micronuclear test). It was shown that epirubicin induced cytogenetic disturbances in the hemopoietic cells within the first 72 hours. The antibiotic had a marked reversible effect on the erythroid population and lymphoid tissues and a moderate toxic action on the granulocyte population. The antibiotic did not affect thrombocytopoiesis. The single administrations had a more pronounced and prolonged myelotoxic and lymphotropic effect.     

rRNA-protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex. Probing by bifunctional Pt(II)-containing reagent

The cleavable homobifunctional reagent dichloro[N,N,N',N'-tetrakis(2-aminoethyl)-1,6-hexamethylenediamminedi platinum (II)] dichloride was used for studying rRNA-protein cross-links in free 35S-labelled 70 S ribosomes and within initiation complex ribosome.AUGU6.fMet-tRNA(fMet). It was shown that the sets of proteins cross-linked to 16 S and 23 S rRNA in free 70 S ribosomes and in 70 S initiation complex do not differ significantly. The authors are the first to demonstrate most of the 23 S rRNA-protein cross-links and some 16 S rRNA-protein cross-links, in particular those with L7/L12 protein.     

Selective alkylation of G24 residue in tRNAPhe

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of oligonucleotide d(pAACCA) was studied. G24 residue located near the sequence C17GGDA21 partially complementary to the oligonucleotide moiety of the reagent was shown to be alkylated. Oligonucleotide d(pAACCA) inhibited the alkylation. Association constant of oligonucleotide derivative with tRNAPhe (10(3) M-1) was evaluated from the dependence of the extent of tRNA modification on the concentration of the reagent. The reported method for selective alkylation of tRNA may be used for preparing photoaffinity derivatives of tRNA bearing an arylazidogroups in desired position.     

Structure of the chromatin with long linker DNA

The method of velocity sedimentation have been used to investigate ionic-strength-induced compaction of sea urchin sperm chromatin characterized by extremely long linker DNA (100 b.p.). The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain have been studied in the range of ionic strength from 0.005 to 0.085. Analysis of these data indicates that such structural parameters of sea urchin sperm chromatin fibre as the diameter of the chain and the length of the chain per nucleosome are quite similar to those of chromatin with shorter linker DNA, but the DNA packing ratio is higher. The structure of sea urchin sperm oligonucleosomes agrees well with the model of three-dimensional zig-zag-shaped chain with linker DNA forming a loop. The possible role of alpha-helical regions of the C-terminal domain of sea urchin sperm histone H1 in the long linker DNA folding is discussed.     

Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)aminobenzylidene]m derivatives of oligodeoxyribonucleotides. IV. Determination of binding sites of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA

By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(pTTTGCTCCCC)rA (IV) (reagents (II)--(IV] followed by the RNase H treatment a number of 16S rRNA fragments have been obtained. Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E. coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA. Good correlation is found between estimated stability of non-perfect 16S rRNA.oligodeoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide. With high concentration of the reagents (II)--(IV) ((2-5) x 10(-5) M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents. It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini. Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.