A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoRI. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 x 106 daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of rdeterminants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: rdeterminants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is:rA-D-C-E-F-B-H-I-L-K-G-M-J1.The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.

The bakers' yeast Saccharomyces cerevsiae is a facultative anaerobe, tolerant to mutations in its mitochondrial genome. Individual cytoplasmic petite mutants retain genetic information derived from any portion of the parental mtDNA, prompting questions concerning distribution of the DNA replication origin(s) on the yeast mitochondrial genome. The experiments described in this paper were designed to test the possibility of using high-frequency yeast transformation as a selection for yeast mtDNA sequences conferring autonomously replicating function. A complete petite mitochondrial genome was inserted into the yeast vector YIp5, and the hybrid plasmid (YRMpl) was used to transform yeast. YRMpl promoted high-frequency transformation ofboth wild-type yeast cells and petite mutant hosts lacking mtDNA and was maintained in each of these strains as a highcopy-number extrachromosomal element. The stability and copynumber properties of YRMpl are similar to those of YRp12, a recombinant plasmid containing a yeast chromosomal autonomously replicating sequence. sequence (ars) are able to transform yeast at high frequency, a readily selectable property (10). This rationale has been used to isolate putative DNA replication origins from yeast chromosomes (11,12) and from a wide variety of heterologous eukaryotic genomes (13).The experiments described here were undertaken to isolate and map yeast mtDNA sequences that are ars elements as assayed by high-frequency yeast transformation. Since many individual petite mtDNAs exist in vivo as amplified tandemly repeated segments (2), it was anticipated that the repetitive nature of these mitochondrial genomes might provide an enriched source ofDNA replication origins. We have shown that a cloned intact petite genome is capable of conferring ars characteristics to a nonreplicating vector and that ars properties of this cloned segment, such as mitotic stability and copy number, are similar to those of the chromosomal DNA sequence TRPl-arsl (7,8,10 Ag of DNA) as a result of stable integration into host cell chromosomal DNA. A few yeast sequences transform at high frequency (500-20,000 transformants per ,g of DNA) and are maintained within the cell as extrachromosomal elements. These properties result from the presence on the transforming plasmid of a DNA sequence conferring autonomously replicating functions (7-9). Hybrid molecules that combine a low-frequency transforming plasmid with an autonomously replicating MATERIALS AND METHODS Strains and Culture Conditions. The p-petite mutant A17-10 was isolated after ethidium bromide mutagenesis of S. cerevisiae strain A10 (a his ade p+). JHC8-24C (a ura3-52 his3-11 his3-15 leu2-3 leu2-112 inol -13 ino4-8 p+) and an ethidium bromide-induced p0 derivative served as hosts in yeast transformation experiments. Bacterial cloning was carried out in a thymine-requiring derivative ofEscherichia coli HB101 (14). Yeast culture media were used as described (15) Rapid Yeast DNA Preparation. High-molecular-weight total cellular DNA was prepared fr...

The size and degree of homogeneity of the repetitive units in purified ribosomal DNA (gamma DNA) from Saccharomyces cerevisiae have been analyzed by restriction endonuclease digestion and heteroduplex mapping. Digestion of the gamma DNA with EcoRI yields seven fragments, digestion with Hind II+III yields five fragments, digestion with Hind III alone yields two fragments, and digestion with Sma I yields one fragment. The sum of the fragment molecular weights after digestion with each of the endonucleases is 5.5-5.6 x 10(6). When the DNA strands of the Sma I fragment are dissociated and reannealed, only homoduplexes are formed. We have concluded from these results that the repeating units in yeast ribosomal DNA are 5.6 x 10(6) datons and are homogeneous in size and composition.

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We have constructed a strain of Saccharomyces cerevisiae that expresses two different members of the multigene family encoding phaseolin, the major seed storage glycoprotein from the French bean, Phaseolus vulgaris. Yeast vector plasmids have been engineered to include a Phaseolus DNA segment that contains the natural 5' and 3' plant genomic regulatory sequences flanking a cDNA copy of the proteinencoding region. Characterization of phaseolin transcripts isolated from transformed yeast cells revealed the presence of two classes of polyadenylylated RNA, approximately 1400 and 1800 bases, which initiate and terminate in plant DNA sequences. Protein extracts from transformants contain phaseolin-immunoreactive proteins similar in size to those isolated from plant tissue. These polypeptides are glycosylated in yeast and their molecular weights are consistent with the possibility that the phaseolin signal peptide has been cleaved to form the mature protein.

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We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PH05) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

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