Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid.

We have developed an effective method to delete or invert a chromosomal segment and to create reciprocal recombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specific recombination system of pSR1, a circular cryptic DNA plasmid resembling 2 microns DNA of S. cerevisiae but originating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing the specific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or two different chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with a plasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placed downstream of the GAL1 promoter. When the transformants were cultivated in galactose medium, the recombination enzyme produced by expression of the R gene created the modified chromosome(s) by recombination between two specific recombination sites inserted on the chromosome(s).     

Chemical modification of neamine

The aminocyclitol antibiotic neamine has been modified by changing the configuration of one or two hydroxyl groups of the aminocyclitol moiety to elucidate the relationship between configuration and antimicrobial activity. 5-Epi-, 6-epi-, and 5,6-diepineamine have been prepared and their antimicrobial activity has been determined against several micro-organisms.     

Chemical modification of aminocyclitol antibiotics

The aminocyclitol antibiotic neamine has been chemically modified at the hydroxyl group on C-6 of the 2-deoxystreptamine moiety. The partially acetylated neamine derivatives, 6,3′,4′-tri-O-acetyl- (3) and 5,3′,4′-tri-O-acetyl-1,3,2′,6′-tetra-N-(ethoxycarbonyl)neamine (4), were prepared by random hydrolysis of the 5,6-O-ethoxyethylidene derivative (2), followed by chromatographic purification. Condensation of 4 and 2,3,5-tri-O-benzoyl-d-ribofuranosyl chloride led to 6-O-(β-d-ribofuranosyl)neamine (7). Analogous condensation of 4 with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide afforded the corresponding 6-O-(d-hexopyranosyl)neamines.     

Guanosine 3':5'-monophosphate-dependent protein kinase from bovine cerebellum. Purification and characterization.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was assayed with calf thymus histone as substrate and partially purified from the soluble fraction of bovine cerebellum. The enzyme was selectively activated by cyclic GMP at lower concentrations; the Ka value for cyclic GMP was 1.7 times 10- minus 8 M whereas that for adenosine 3':5'-monophosphate (cyclic AMP) was 1.0 times 10- minus 6 M. The Km value for ATP was 1.0 times 10- minus 5 M. A high concentration of Mg-2+ (100 mM) was needed for maximum stimulation by cyclic GMP and maximum reaction rate. The pH optimum was 7.5 to 8.0. The isoelectric point was pH 5.7. The molecular weight was about 140,000 as estimated by gel filtration. The enzyme was unable to activate muscle glycogen phosphorylase kinase, and was clearly distinguishable from cyclic AMP-dependent protein kinase in kinetic and catalytic properties. Comparative data on cyclic GMP-dependent and cyclic AMP-dependent protein kinases in this tissue are presented.     

The synergetic enzyme theory of muscular contraction: II Relation between Hill's equations and functions of two-headed myosin.

Based on the assumption of nonidentical two heads of myosin it is pointed out that a strong motive force is generated in actomyosin pair only when ATP-decomposition occurs co-operatively at the both heads and that the tension-independent part of shortening heat is liberated when an ATP molecule is decomposed only at the burst head. These two actions of actomyosin pair are related to the two states of force-generator in Huxley-Simmons' model. Elementary cycles at different positions in a sarcomere are interacted each other through feedback loop via sliding motion of muscular filaments. Due to this synergetic interaction the rate constant for the rate-determining step of elementary cycle has a dependence on velocity v of shortening such as k = k ° + κv. From these functions and properties of actomyosin system in vivo, the following properties of muscle are explained consistently in a quantitative manner: (1) Hill's equation on the relationship between tension and velocity of shortening, (2) damped oscillations in tension and in muscular length around steady state, (3) Hill's energy equation improved in 1964, (4) the chemical equivalence of shortening heat, (5) the influence of tension on the incorporation of radio-active phosphate into ATP and (6) the asymmetric activation by actomyosin system only for the forward reaction, the decomposition of ATP.     

Temperature-sensitive virus derived from BHK cells persistently infected with HVJ (Sendai virus).

BHK-HVJ cells, a cell line of baby hamster kidney cells persistantly infected with HVJ (Sendai virus), started to produce infectious virus by shifting down the incubation temperature from 38 to 32 C. The virus derived from BHK-HVJ cells, designated as HJV-pB, was effectively neutralized with antibody against wild-type virus (HVJ-W) which was used for the establishment of BHK-HVJ cells. HVJ-pB replicated in eggs at 32 C, but not at 38 C, while HVJ-W grew equally well at both temperatures. When BHK cells infected with HVJ-PB were incubated at 38 C, production of infectious virus, hemagglutinin, and neuraminidase was markedly restrained, whereas a considerable amount of viral nucleocapisid and envelope antigens was detected in the cells by complement fixation tests. These viral activities became detectable immediately after temperature shift-down from 38 to 32 C even at the later stage of infection. HVJ-pB was indistinguishable from HJV-W with respect to particle size, density, and morphological characteristics, but appeared to possess a higher neuraminidase activity and was inactivated more rapidly at 50 C than HVJ-W. HVJ-pB was less cytocidal and could easily cause latent infection in BHK and mouse L cells.