The induction of multinuclearity in agitated and in aging cultures of Acanthamoeba sp. Neff

Partially synchronized secondary cultures of chicken embryo fibroblasts were exposed to Rous sarcoma virus (RSV) at various times in the cell cycle. The initiation of normal virus production could be inhibited by treatment with excess thymidine or with cytosine arabinoside without any effect on mitosis. This result is consistent with the hypothesis that the provirus of RSV is DNA, although the virion of RSV contains only RNA. It was, also, found that treatment which prevented or interferred with the first mitosis after exposure to virus prevented the initiation of normal virus production. Later mitoses did not appear to be necessary. A corollary of this finding is that virus production is synchronized by mitosis and that the length of the latent period depends upon when in the cell cycle a cell is exposed to virus.     

Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen")

Lankford, C. E. (The University of Texas, Austin), James R. Walker, James B. Reeves, N. H. Nabbut, B. R. Byers, and R. J. Jones. Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen"). J. Bacteriol. 91:1070-1079. 1966.-When cells of Bacillus megaterium, grown on Brain Heart Infusion Agar, were inoculated into a chemically defined medium, they exhibited a division lag which was an inverse function of inoculum size. The addition of filtrates of cultures from the same medium eliminated the inoculum-dependent component of lag, but not an inoculum-independent residual lag of constant duration. Culture filtrate of B. subtilis var. niger not only eliminated its inoculum-dependent lag but also was required to sustain exponential division. Dose-response and "growth time" bioassays, developed to measure lag-reducing activity of filtrates, demonstrated accumulation of active filtrate factor to a "critical" concentration prior to division initiation. Addition of this concentration to cultures eliminated the inoculum-dependent lag. Accumulation of the factor ceased temporarily at onset of division, but excretion was resumed later during exponential growth. Accumulation of a lag-reducing, cell-associated factor followed a similar course. Chromatographic and bioautographic analyses of culture filtrates of B. megaterium indicated that a single substance was primarily responsible for their activity. Results of dose-response tests for reciprocal activities of filtrates of different Bacillus species and strains suggested production of different factors by some, and of different quantities of similar factors by others. It is proposed that such endogenous factors which are synthesized and accumulate to a population-dependent concentration as a requisite to initiation and maintenance of division be designated as "schizokinens."     

The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo.

The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.     

Insertional Mutations in the Yeast Hop1 Gene: Evidence for Multimeric Assembly in Meiosis

The HOP1 gene of Saccharomyces cerevisiae has been shown to play an important role in meiotic synapsis. In this study we analyzed the mechanism of this function by phenotypic characterization of novel in-frame linker-insertion mutations located at various sites throughout the HOP1 coding sequence. Among 12 mutations found to cause defects in meiotic recombination and spore viability, three were temperature-sensitive for the spore viability defect. Although substantial meiotic recombination was found for these conditional alleles at the restrictive temperature, the level of exchange measured in spo13 meiosis was reduced in some of the monitored intervals, indicating that nondisjunction resulting from a deficit in crossing over could account for SPO13 spore inviability. Intragenic complementation between linker-insertion alleles was assessed by testing the viability of spores generated from heteroallelic diploids after SPO13 meiosis. Complex patterns of complementation and enhancement of the spore-inviability phenotype indicate that HOP1 functions in a multimeric complex. In addition, the ability of alleles which map near the carboxyl terminus to complement several other alleles provides evidence for a functional domain in this region of the protein. Two previously identified multicopy suppressors of the conditional hop1-628(ts) allele were tested for their effects in cells bearing the linker-insertion hop1 alleles. Overexpression of REC104 from a 2μ plasmid was shown to enhance the spore viability of every allele tested, including a hop1 disruption allele. On the other hand, suppression by overexpression of RED1 from a 2μ plasmid was found only for allele hop1-628(ts). Surprisingly, similar overexpression of RED1 in strains bearing several other conditional hop1 linker-insertion alleles caused enhanced spore lethality. This finding, in conjunction with the evidence for a carboxy-terminal domain, provides new insight into the nature of interactions between the HOP1 and RED1 products in meiosis.     

Human β-glucuronidase: Assignment of the structural gene to chromosome 7 using somatic cell hybrids

-Glucuronidase (GUS) has become an important enzyme model for the genetic study of molecular disease, enzyme realization, and therapy, and for the biogenesis and function of the lysosome and lysosomal enzymes. The genetics of human -glucuronidase was investigated utilizing 188 primary man-mouse and man-Chinese hamster somatic cell hybrids segregating human chromosomes. Cell hybrids were derived from 16 different fusion experiments involving cells from ten different and unrelated individuals and six different rodent cell lines. The genetic relationship of GUS to 28 enzyme markers representing 19 linkage groups was determined, and chromosome studies on selected cell hybrids were performed. The evidence indicates that the -glucuronidase gene is assigned to chromosome 7 in man. Comparative linkage data in man and mouse indicate that the structural gene GUS is located in a region on chromosome 7 that has remained conserved during evolution. Involvement of other chromosomes whose genes may be important in the final expression of GUS was not observed. A tetrameric structure of human -glucuronidase was demonstrated by the formation of three heteropolymers migrating between the human and mouse molecular forms in chromosome 7 positive cell hybrids. Linkage of GUS to other lysosomal enzyme genes was investigated. -Hexosaminidase HEX _B) was assigned to chromosome 5; acid phosphatase₂ (ACP ₂) and esterase A₄ (ES-A ₄) were assigned to chromosome 11; HEX _A was not linked to GUS; and -galactosidase (-GAL) was localized on the X chromosome. These assignments are consistent with previous reports. Evidence was not obtained for a cluster of lysosomal enzyme structural genes. In demonstrating that GUS was not assigned to chromosome 9 utilizing an X/9 translocation segregating in cell hybrids, the gene coding for human adenylate kinase₁ was confirmed to be located on chromosome 9.Supported by NIH Grants HD 05196, GM 20454, and GM 06321, by NSF Grant BMS 73-07072, and by HEW Maternal and Child Health Service, Project 417.     

Blockade of alpha-adrenergic receptors by analogues of phosphatidylcholine

The experimental evidence reviewed in this article suggests that the kidneys may have an additional function in regulating blood pressure besides their role in controlling both blood volume by urine formation and the relative state of vasoconstriction by the renin-angiotensin system. That is, the kidneys may have an additional influence upon the vasculature of a hormonal vasodilating system. The interstitial cells of the renal medulla appear to be mediating this activity and lipid compounds have been extracted from the renal medulla which display depressor activity. One such compound, the antihypertensive polar renomedullary lipid (APRL), has been demonstrated to consist of specific alkyl ether analogues of phosphatidylcholine. The vascular responses to these compounds include vasodilation of both arterioles and venules, rapid lowering of arterial blood pressure with little or no tachycardia, increased depressor activity in hypertensive animals, and blockade of vascular smooth muscle alpha 1-adrenergic receptors. Most recently, APRL and a synthetic analogue, 1-0-octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine, have been used to demonstrate alpha-adrenergic receptor blockade on a smooth muscle cell line (DDT1) by radioligand assays. This action may be due to the insertion of these compounds into cell membranes causing subsequent steric interactions and blockade of the alpha-adrenergic receptor.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Multiexon deletion in the procollagen III gene is associated with mild Ehlers-Danlos syndrome type IV

We have characterized a deletion of approximately 9 kilobases which spans from intron 33 to exon 48 of one pro-alpha 1 (III) collagen allele in a patient with Ehlers-Danlos syndrome type IV. The mutation results in the production of an in-frame species of mRNA which lacks the sequences corresponding to residues 595-1,008 of the triple-helical domain. Thus, half of the pro-alpha 1 (III) chains synthesized by the patient's fibroblasts are nearly 30% shorter than normal. The procollagen III molecules composed of either three normal length or three shortened chains are thermally stable and efficiently secreted. In contrast, the procollagen III molecules that contain one or two shortened chains are unstable and are not secreted. Failure to secrete unstable molecules and a residual functional role of the shortened but stable homotrimers may explain the somewhat milder phenotype of this individual compared with that of another Ehlers-Danlos type IV patient bearing a deletion of similar size in the amino-terminal portion of the alpha 1 (III) collagen chain.