Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2).

A physical map of the chromosome of Streptomyces lividans 66 ZX7 was constructed by ordering the macrorestriction fragments generated from the genomic DNA with the restriction enzymes AseI and DraI. AseI and DraI linking cosmids (i.e., recombinant cosmids including either AseI or DraI sites) were isolated from a gene bank and used as hybridization probes against Southern transfers of pulsed-field gel electrophoresis (PFGE) restriction patterns. The DraI sites were precisely mapped by PFGE analyses of AseI-DraI double digests and hybridization with the AseI junctions. The 16 AseI and 7 DraI fragments were aligned as a single chromosome of about 8,000 kb. The data supported the interpretation that the chromosome is a linear structure. The related strain Streptomyces coelicolor A3(2) M145, recently mapped by H. Kieser, T. Kieser, and D. A. Hopwood (J. Bacteriol. 174:5496-5507, 1992), was compared with S. lividans at the level of the genomic structure by hybridizing the linking cosmids to Southern transfers of PFGE patterns. In spite of little apparent similarity in their restriction patterns, the comparison of the physical maps revealed a common structure with an identical ordering of the cosmid sequences. This conservation of the map order was further confirmed by assigning genetic markers (i.e., cloned genes and DNA elements relevant to the unstable region) to the AseI fragments.     

Random insertion of Tn4560 in Streptomyces lividans and Streptomyces avermitilis

Summary Pulsed-field gel electrophoresis (PFGE) was used to show that insertions of transposon Tn4560 in Streptomyces lividans 66 and the avermectin-producer S. avermitilis are randomly distributed. DNA from the latter strain suffered degradation during electrophoresis that could be avoided by modification of the buffer. Inverse PCR primers were developed for Tn4560.     

Physical Characterization of Plasmid pMQV10 from a Steroid Biotransforming Strain of Micrococcus

The presence of a single plasmid 8.5 kb in size has been demonstrated in a cholesterol biotransforming strain of Micrococcus. A detailed physical map of the plasmid has been constructed using various restriction enzymes. Streptomycin resistance has been localized on a 1.8-kb fragment of pMQV10.     

Glycation of Brain Actin in Experimental Diabetes

Abstract: Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose-6-phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G-actin to form F-actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet-derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.     

Reproductive biology of Carcharhinus acronotus in the coastal waters of South Carolina

The reproductive biology of blacknose sharks Carcharhinus acronotus in the western North Atlantic Ocean was studied by examining specimens collected in the coastal waters of South Carolina. Males begin the maturation process between 875 and 910 mm fork length ( L _F), as indicated by the presence of functional claspers and siphon sacs. The presence of vitellogenic oocytes and developing oviducal glands and uteri indicated that females begin to mature at c . 870 mm L _F. Length at which 50% of the population reached maturity was 896 and 964 mm L _F, equivalent to 4·3 and 4·5 years, for males and females, respectively. Gonado‐somatic indices suggested that spermatogenesis and vitellogenesis began after December. Mating took place during the end of May and the beginning of June. Fertilization occurred during late June and early July, suggesting that female blacknose sharks were capable of sperm storage. Based on the timing of fertilization and occurrence of females carrying near‐term pups in late May and early June, the gestation period for blacknose sharks was c . 11 months. Female blacknose sharks reproduced biennially based on the absence of vitellogenic oocytes in near‐term females and there being no indication of vitellogenesis in postpartum females. Male blacknose sharks were capable of reproducing annually as indicated by turgid genital ducts, which were observed in all mature males collected during late May and early June.     

Structure of an amplifiable DNA sequence in Streptomyces lividans 66

Summary Spontaneous chloramphenicol-sensitive mutants of Streptomyces lividans 66 had previously been shown to be very unstable and to yield arginine auxotrophic mutants at a frequency of 25% of spores; the Arg^- mutants had amplified a particular 5.7 kb DNA sequence to over one hundred tandem copies per genome. In this paper we report the cloning of the amplifiable region from amplified and wild-type strains. This showed that the amplifiable fragment is already present as a duplication in wild type cells. Hybridisation experiments also demonstrated that in the amplified strains there was a deletion of neighbouring DNA sequences to one side of the amplifiable element; sequences to the other side remain intact.     

Genetic instability inStreptomyces

Genetic instability is very common amongStreptomyces strains and the affected strains display several distinctive phenotypes. In several cases DNA rearrangements, specifically deletions and amplifications of specific segments of DNA, were demonstrated. Depending upon the reproducible amplification of a segment in independent isolates one could predict the basic structural elements involved in the amplification process. In the case ofS. lividans 66,5.7 kb amplifiable DNA sequence is located near the end of the linear chromosome. Amplification of this sequence and deletion of the chromosome linked to it leads to the creation of a new end or in some cases even circularisation of the chromosome occurs. A model incorporating these aspects is discussed. The possible involvement of proteins encoded by the amplifiable region is also discussed.     

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome

Thirty-two 2-deoxygalactose-resistant mutants with DNA amplifications were isolated from Streptomyces lividans 66 strains carrying plasmid pMT664, which carries an agarase gene (dagA) and IS466. Thirty-one of the mutants carried amplified DNA sequences from a 70 kb region about 300 kb from one end of the linear chromosome in this species. In 28 of the mutants, all the wild-type sequences between the amplified region and the start of the 30 kb inverted repeat that forms the chromosome end were deleted. Thus, there appeared to be loss of one chromosome end and its replacement by the DNA amplification. In some mutants there amplification of a previously characterised 5.7 kb sequence that lies about 600 kb from the other chromosome end was also noted.     

Construction of Tn5424 — A new transposon forStreptomyces

Summary Tn5424 contains one complete copy of IS466 and 0.64 kb end fragment of IS466 flanking a thiostrepton-resistance (isr) marker gene. In contrast to an earlier IS466-transposon, Tn5424 does not show any structural rearrangements during integration into the host chromosome. Further investigations by pulsed-field gel electrophoresis indicated a random insertion of the element in the chromosome ofStreptomyces lividans 66 ZX7.     

Liver Transplantation in Man—II,a Report of two Orthotopic Liver Transplants in Adult Recipients

Two patients with primary hepatic malignancy were treated by hepatectomy and orthotopic liver transplantation. In both cases the donor liver was infused with cold solutions and kept chilled without continuous perfusion. There was immediate satisfactory hepatic function in both transplants.The first patient died after 11 weeks from overwhelming bacterial and fungal infections probably secondary to hepatic infarction due to thrombosis of the recipient hepatic artery. The thrombus occurred at the site of the arterial clamp. In an attempt to control the growth before transplantation, the patient had been treated with large doses of chlorambucil, which resulted in extreme marrow depression and septicaemia.The second patient developed cholestatic jaundice during the second and third weeks after transplantation, with histological evidence of mild rejection, which was controlled by increasing the dose of immunosuppressive agents. He is now well, having returned to work six weeks after the operation.Though the first patient showed no evidence of rejection, it is concluded that patients receiving liver allografts should receive immunosuppressive therapy.