Transformation of Penicillium chrysogenum by a vector containing a mitochondrial origin of replication

Summary In order to establish a transformation system for P. chrysogenum autonomously replicating vectors were constructed using mitochondrial DNA sequences from the fungus. A physical map of the mt DNA of a production strain was established using ten different restriction enzymes. Unexpectedly, the mt DNA of this strain proved to be significantly smaller than that of a second strain from a culture collection (27 kb versus 49 kb). Various fragments representing about 71% of the 27 kb mt DNA were cloned and, at first, preselected for replicating activity in an intermediate host (Saccharomyces cerevisiae). Two of these fragments also promoted autonomous replication in P. chrysogenum, which was confirmed by isolation of bulk DNA and transfer into E. coli. For selection of transformants in P. chrysogenum the prokaryotic kanamycin resistance gene was used which increased about twofold the resistance against G418. Present address: Institut für Biotechnologie, Fachgebiet Mikrobiologie, Techn. Universität Berlin, Seestr. 13, D-1000 Berlin 65     

Evidence that the normal route of replication-allowed Red-mediated recombination involves double-chain ends.

Recombination mediated by the Red pathway of bacteriophage lambda is focused towards sites of double-chain cuts. Double-chain ends created either by type II restriction enzymes acting at unmodified recognition sites or by lambda's packaging enzyme, terminase, acting at cos are utilized in a manner similar to the double-chain break repair pathway of recombination in yeast. When lambda is allowed to recombine during replicative growth, spontaneous recombination is approximately evenly distributed along the chromosome. It has been proposed that replication-allowed recombination also is initiated by double-chain ends. In order to test this hypothesis we ask if the in vivo expression of the Mu gam protein is inhibitory to Red recombination. Mu gam has been shown in vitro to bind to linearized duplex DNA and to shield bound DNA from exonucleases. The expression of Mu gam is found to be inhibitory to Red recombination whether replication is blocked or allowed. As a control we ask if Mu gam inhibits Int-mediated recombination. It has been well documented that the Int pathway of recombination does not involve any double-chain breaks and, consistent with this, the Int pathway is not inhibited by Mu gam. We suggest that the in vivo expression of Mu gam or other similar activities may be a generally useful way to determine if those processes that respond to an artificially introduced double-chain cut normally involve double-chain ends.     

Campylobacter pylori, the spiral bacterium associated with human gastritis, is not a true Campylobacter sp.

Comparison of partial 16S rRNA sequences from representative Campylobacter species indicates that the Campylobacter species form a previously undescribed basic eubacterial group, which is related to the other major groups only by very deep branching. This analysis was extended to include the spiral bacterium associated with human gastritis, Campylobacter pylori (formerly Campylobacter pyloridis). The distance between C. pylori and the other Campylobacter species is sufficient to exclude the pyloric organism from the Campylobacter genus. The results indicate that C. pylori is more closely related to Wolinella succinogenes than it is to the other Campylobacter species inspected. Another close relative of the campylobacters was found to be Thiovulum, a sulfide-dependent marine bacterium.     

The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene (pho A) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A. In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.     

In vivo depletion of mannose receptor bearing cells from rat liver by ricin A chain: effects on clearance of beta-glucuronidase

Ricin A chain has previously been shown to intoxicate macrophages in vitro following binding and endocytosis by the macrophage mannose receptor. In this report it is demonstrated that the intravenous injection of ricin A chain in nephrectomized rats leads to a prolonged plasma half-life for [125I]beta-glucuronidase, a ligand for the mannose receptor. Clearance of [125I]asialofetuin, a ligand for the galactose receptor of hepatocytes, was unaffected by injection of A chain. Microscopic examination of the livers of A chain-treated animals revealed a loss of phagocytic cells from the liver sinusoids. These results suggest that ricin A chain may be useful as a toxin specific for mannose receptor bearing cells of the reticuloendothelial system.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

Purification of the growth-related protein p25 of the Ehrlich ascites tumor and analysis of its isoforms

1. Two of the three isoforms of the growth-related protein p25 of the Ehrlich ascites tumor have been purified to homogeneity by giant two-dimensional polyacrylamide gel electrophoresis. 2. Antibodies raised against the isoform p25/1 react also with isoforms p25/2 and p25/3. 3. Limited tryptic digestion of p25/1 and p25/2 resulted in similar oligopeptide patterns. Corresponding oligopeptides of both isoforms have identical amino acid sequences. 4. The isoforms p25/2 and p25/3 are phosphorylated derivatives of unphosphorylated p25/1. The phosphorus is bound to serine and a further unknown phosphorylation site.     

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.     

1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements

The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity. The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates. The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C. For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction. The minimum length of the single-stranded tail was determined to be less than 5 nucleotides. Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction. This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase. Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome. The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.