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The gene for a previously unidentified small nuclear RNA has been cloned from Saccharomyces cerevisiae and its nucleotide sequence has been determined. The RNA, snR30, was mapped to a unique coding sequence 605 nucleotides long. SnR30 appears to be one of the most abundant snRNAs of S, cerevisiae in that it can be resolved by ethidium bromide staining on one-dimensional denaturing gels of total yeast RNA. Like other snRNAs, snR30 is enriched in nuclei preparations and possesses a trimethyl guanosine cap structure at its 5' end. After substituting one allele of the wild type gene in a diploid strain for a deleted gene, after sporulation, haploid strains carrying the deletion were unable to grow, indicating that snR30 is required for an essential, but as yet, unknown function. The nucleotide sequence close to the initiation site of the SNR30 gene is similar to that of other yeast SNR genes whose transcripts are associated with pre-rRNA, suggesting that snR30 is related to this group of snRNAs.

snR17, one of the most abundant capped small nuclear RNAs of Saccharomyces cerevisiae, is equivalent to U3 snRNA of other eukaryotes. It is 328 nucleotides in length, 1.5 times as long as other U3 RNAs, but shares significant homology both in nucleotide sequence and in predicted secondary structure. Human scleroderma antiserum specific to nucleolar U3 RNP can enrich snR17 from sonicated yeast nuclear extracts. Unlike other yeast snRNAs which are encoded by single copy genes, snR17 is encoded by two genetically unlinked genes: SNR17A and SNR17B. The RNA snR17A is more abundant than snR17B. Deleting one or other of the genes has no obvious phenotypic effect, except that the steady-state level of snR17B is increased in snr17a- strains. Haploid strains with both genes deleted are inviable, therefore yeast U3 is essential.

Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E. coli. This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase. By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame. A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence. In this step, however, we use an oligonucleotide that has a degenerate sequence. That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence. The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest. We discuss the results obtained by applying the method to the Rop gene.

We have studied eight frameshift mutations induced by the Escherichia coli mutator allele mutD5 in a derivative of the bacteriophage M13mp8, carrying an insertion of 91 base pairs derived from the tetR gene of pBR 322. All mutations were analyzed by the dideoxy sequencing method and were found to be deletions of a GC base pair which occurred in regions characterized by the presence of at least two GC base pairs. We have attempted to explain these results by the looping-out model, which was previously proposed to unify the results obtained with mutD5.

We describe the construction and properties of pEMBLY plasmids. They belong to a new family of yeast shuttle vectors which are derived from plasmid vector pEMBL9 and offer the following improvement: relatively small size; large number of cloning sites; screening for insert-containing plasmids on indicator plates; different combinations of genes which complement auxotrophic deficiencies and sequences that support DNA replication in Saccharomyces cerevisiae; and ability to isolate the plasmid DNA in single-stranded (ss) form. The yeast S. cerevisiae can be efficiently transformed by these plasmids in both the ss and double-stranded (ds) forms. Finally, the presence of the phage f1 intergenic region allows one to obtain the cloned sequences in the ss form upon infection with the wild-type ss phage [Dotto et al., Virology 114 (1981) 463-473].

A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage lambda vector, lambda 1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in lambda 1059 requires deletion of the lambda red and gamma genes. The recombinants are therefore spi- and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage P2. Random fragments suitable for insertion into lambda 1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a. This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because G-A-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved lambda 1059. By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed. DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection.

We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process. Apoferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures. We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding-assembly process is altered or completely impaired. The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the alpha-peptide of beta-galactosidase. In the wild type situation, the alpha-peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function. We show that by selecting for mutations that restore beta-galactosidase activity we are able to identify ferritin mutations that affect the folding-assembly process. The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy-terminal E helix in the 4-fold axis.

Human H-chain ferritins bearing sequence changes in the 3-fold channels have been expressed in E. coli to investigate the role of these channels in iron-storage processes. The proteins assemble into shells resembling those of native ferritins. Iron uptake measurements indicate that residues in the 3-fold channels are involved neither in initial Fe(II)-oxidation nor in iron-core nucleation.

In this paper we describe a method to select base pair substitution mutants constructed in vitro. The mutagenesis is performed by forcing mistakes during in vitro synthesis from a primer annealed to a single stranded DNA template. The selection is based on the fact that, following transformation, the progeny of the strand elongated in vitro and the template strand have different phenotypes. The method is general and applicable to any DNA segment; the type of base pair substitution and its position can be chosen at will. The combined efficiency of mutagenesis and selection allows for 85% frequency of mutants in all analyzed clones.

Derivatives of the yeast 2mu plasmid with the cis-acting locus STB (also called REP3) are stably maintained if two plasmid-encoded proteins are present in trans. There are conflicting reports of both the extent of STB and its possible involvement in plasmid partition or copy number control. We have resolved the controversy by constructing 2micro derivatives with a conditional STB function, and showing that when STB is inactivated plasmids become concentrated in a small fraction of the population although the total number of plasmids remains unaltered. Moreover we show that STB consists of two functionally distinct domains which we call STB-proximal and STB-distal relative to the origin of replication. Although STB-proximal is sufficient for proper partitioning, this function is severely disrupted by active transcription from neighbouring sequences. STB-distal is important to protect STB-proximal and ORI from such transcription, and can be effeciently replaced by a 94-bp terminator fragment in an orientation-dependent manner. We find that STB-distal contains an additional element which depresses transcription from upstream promoters. We also describe the phenomenon of replicaton inhibition which we believe can exlain the anomalous instability of some yeast plasmids.

The endogenous 2 mu plasmid of yeast encodes a site-specific recombinase FLP that can cause an amplification of plasmid copy number. Using strains overexpressing 2 mu plasmid proteins from chromosomal constructs to disrupt the normal balance of gene products, we show here that copy number is controlled by regulating the transcript level of FLP. Expression of FLP is negatively regulated over a 100-fold range by the joint action of the plasmid-encoded REP1 and REP2 proteins, which also have a role in plasmid partition. We also show that the product of the fourth plasmid open reading frame D increases FLP expression by relieving the repression caused by REP1 and REP2. This is the first demonstration of a function for this gene, which we call RAF. The transcription of RAF is also repressed by REP1 and REP2 acting together, but requires a higher level for complete inhibition than that required to repress FLP. Copy number is therefore negatively regulated by REP1-REP2 concentration both by direct repression of FLP and indirectly, by control of the positive element, the anti-repressor RAF. We propose that these antagonistic regulatory mechanisms amplify the signal produced by a small fall in copy number.

We show that a protein of 63 amino acids is the product of the rop gene, a gene which negatively regulates the copy number of plasmids of the ColE1 family. Rop protein purified to homogeneity inhibits ColE1 plasmid replication in a bacterial extract. Furthermore, we show that Rop inhibition requires RNA I. In a purified in vitro system that can support primer transcription and processing, Rop affects primer formation in two ways: first, it elicits transcription termination oat nucleotide 220, and second, it increases the ability of RNA I to inhibit RNAase H processing of the primer. The analysis of these data and the comparison with the results obtained in vivo with transcription fusion experiments allow us to propose a tentative model of the molecular mechanism underlying Rop-RNA I inhibition.

To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H). As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction. The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e. at pH 7 and in presence of iron core. We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus. These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation. The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core. It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.

We have studied the interactions between the three molecules Rop, RNAI and RNAII that are involved in the regulatory mechanism controlling the replication of ColE1 plasmids. We show that it is possible to purify the two RNA molecules by passing an RNA mixture through an affinity column containing Rop immobilized to a solid support. The dissociation constants of the Rop-RNAI and Rop-RNAII complexes are of the order of 10(-4) M, several orders of magnitude higher than dissociation constants of stable protein-nucleic acid complexes (10(-10) M in the lambda repressor system). Although complete RNAI molecules have higher affinity, stem-and-loop I alone can also bind Rop, suggesting that this structure plays an important role in the interaction. Rop protects the stems of RNAI and RNAII from digestion by RNases while the sensitivity of the loops to digestion by RNase T1 is not affected by high concentrations of Rop. We propose a model for Rop-RNAI/RNAII interaction in which the dimeric protein acts as an adaptor between stem structures to position the two RNAs in the correct position for loop interaction.

Preliminary crystallographic data are given for Rop, a protein involved in the control of replication of plasmids of the ColE1 family.

Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that controls the copy number of plasmid of the ColE1 family by increasing the affinity between two complementary RNAs. The Rop dimer is a bundle of four tightly packed alpha-helices that are held together by hydrophobic interactions. We have systematically altered, by site directed mutagenesis, most of the solvent exposed amino acids of the Rop bundle and we have identified the alterations that cause a decrease of the activity of the regulatory molecule. We conclude that Rop folding is rather insensitive to amino acid substitutions and to other mutations as drastic as deletions and insertions. Looking along the 2-fold symmetry axis the amino acid side chains whose alterations affect the function of Rop are all located on one side of the molecule. Furthermore they are clustered at the extremities of the alpha-helix bundle, the only exception being the aromatic ring of Phe-14.

Previous experiments have shown that mutations in the Ai gene can suppress the growth defect of lambda N- phages. Many temperature resistant derivatives of phage lambdatsN9 have been isolated and among these 5 have been found which are Ai- and have an amber suppressible behaviour. These mutants can help in defining the role of the Ai gene in phage lambda development.

We have constructed a series of plasmids, the pEMBL family, characterized by the presence of 1) the bla gene as selectable marker, 2) a short segment coding for the alpha-peptide of beta-galactosidase and containing a multiple cloning sites polylinker, 3) the intragenic region of phage F1. pEMBL plasmids have the property of being encapsidated as single stranded DNA, upon superinfection with phage F1. These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used. However, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler. In addition, and most importantly, long inserts have a higher stability in pEMBL plasmids than M13 vectors.

We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2. The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins. All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified. We have shown that the ferritin synthesized in E. coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.

A mathematical model for the interaction kinetic in phage-bacterium culture is proposed. An appropriate analytical relationship between phage and bacterial concentrations has been derived. Characteristic kinetic constants have been obtained by comparing the experimental growth curves with computer-simulation analysis. The model allows also to evaluate the phage-yield as a function of the initial concentrations.

The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides. By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA. The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription. The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scR1). ScR1 is neither 3'-polyadenylated nor 5'-trimethylguanosine capped. We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region. Haploid strains carrying the scr1-delta lesion grew very slowly, segregated cytoplasmic petites [( rho-]) at high frequency, and showed signs of aberrant cell division. A secondary structure model for scR1 shows some of the conserved features of the signal recognition particle 7SL RNAs.