Genes coding for 5S ribosomal RNA of the nematode Caenorhabditis elegans

We have identified a 1-kb genomic sequence that represents the major class of 5S rRNA genes in the nematode Caenorhabditis elegans. This 1-kb sequence is tandemly repeated 110 times in the haploid genome forming a single homogeneous gene family. Other nematode genomic sequences, distinct from the major 1-kb repeat class but homologous to it, may represent dispersed 5S rRNA genes or the ends of a gene cluster. One such fragment shows a restriction fragment length difference between two C. elegans strains. This should allow the genetic analysis of 5S rRNA-coding DNA (5S X rDNA) and its flanking regions in C. elegans.     

What makes wild rabbits drink?

Wild rabbits Oryctolagus cuniculus (L) introduced to Australia over a century ago successfully colonized diverse environments in a large part of the continent varying from arid desert, alps, to lush grasslands and coastline where water and salt may be either abundant or very scarce. Wild rabbits caught in Northern Victoria were studied under laboratory conditions, where they adapted to dry pelleted food and drank regularly water and a cafeteria of electrolyte solutions offered. Intracerebroventricular (IVT) infusion of angiotensin II (AII) in doses 10, 50 and 500 ng/h did not increase their water drinking, but increased salt appetite, although it was delayed one or more days after the beginning of AII infusion. IVT infusion of AII 500 ng/h for one day caused a halving in water intake and a tenfold increase in sodium excretion. These were followed by compensatory changes in water and 0.5 M NaCl intake on the consecutive days. IVT infusion of AII 50 ng/h for one day induced an increased urinary sodium excretion, a negative sodium balance which was not followed by an increased salt appetite. IVT infusion of AII 10 ng/h for five days caused a progressive increase in sodium excretion and salt appetite which were significant on the fourth day of infusion and both remained eight-ten times greater than control levels for three days after the cessation of infusion. Water intake was unchanged. IVT infusion of 0.3 M Na-CSF for two days reduced water and food intake, and caused a negative sodium balance on the second day of infusion which was not followed by increase in salt appetite.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of IIIGlc of the phosphoenolpyruvate-glucose phosphotransferase system in inducer exclusion in Escherichia coli.

The phosphoenolpyruvate-D-glucose phosphotransferase system of Enterobacteriaceae is thought to regulate the synthesis and activity of a number of catabolite uptake systems, including those for maltose, lactose, and glycerol, via the phosphorylation state of one of its components, IIIGlc. We have investigated the proposal by Kornberg and co-workers (FEBS Lett. 117(Suppl.):K28-K36, 1980) that not IIIGlc, but an unknown protein, the product of the iex gene, is responsible for the exclusion of the above-mentioned compounds from the cell. The iex mutant HK738 of Escherichia coli contains normal amounts of IIIGlc as measured by specific antibodies, in contrast to crr mutants that lack IIIGlc. The IIIGlc of the iex strain functions normally in glucose and methyl alpha-glucoside transport, and the specific activity in in vitro phosphorylation is approximately 60% of that of the parent. The IIIGlc activity of the iex strain is, however, heat labile, in contrast to the parental IIIGlc, suggesting that the mutant contains an altered IIIGlc. This is supported by the observation that IIIGlc from the iex strain cannot bind to the lactose carrier. Thus it cannot inhibit the carrier, and this explains why the uptake of non-phosphotransferase system compounds in an iex strain is resistant to phosphotransferase system sugars. The introduction of a plasmid containing a wild-type crr+ allele into the iex strain restores the iex phenotype to that of the iex+ parent. The IIIGlc produced from the plasmid in the iex strain is heat stable and binds normally to the lactose carrier. These results lead to the conclusion that the iex mutation is most likely allelic with crr and results in an altered, temperature-sensitive IIIGlc that is still able to function D-glucose and methyl alpha-glucoside uptake and phosphorylation and in the activation of adenylate cyclase, but is unable to bind to and inhibit the lactose carrier.     

Phospholipids as dynamic participants in biological processes

Phospholipids are described as active biological molecules. Three distinctly different roles are examined. The first centers on protein-lipid interactions and the lipid requirement expressed by certain enzymes. This category is illustrated by two soluble proteins of the blood coagulation scheme, Factor IXa and Factor Xa, and by an integral membrane protein, the (Ca2+ + Mg2+)-ATPase of human erythrocytes. The next two examples depict phospholipids as active participants in membrane-mediated events. In the first of these, termed the phosphoinositide effect, a phospholipid becomes a substrate during membrane signaling, and its products presumably act as second messengers. In the second example, a phospholipid is a signal that, among other reactions, induces the phosphoinositide effect. Here, the phospholipid (platelet activating factor) serves as a lipid chemical mediator. These examples show that phospholipids behave not only as structural molecules but also as dynamic, functionally important components of cells.     

Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding

[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.     

Peroxidase activity in the mouse uterus, placenta and fetus during pregnancy

Changes in peroxidase activity during pregnancy were examined in CD-1 mice. Peroxidase activity was measured with guaiacol as the substrate in uterine extracts of nonpregnant mice and in uterine, placental, and fetal extracts of pregnant mice on days 9, 12, 14, 16, and 18 of gestation. Uterine peroxidase activity in nonpregnant mice was high, but declined logarithmically to only 0.2% by day 18 of pregnancy. In contrast to this decline, a concomitant 50-fold logarithmic increase in fetal peroxidase activity was observed between day 12 and 18. Activity in placental extracts did not change significantly throughout the gestational period examined. These results suggest that membrane bound peroxidase in mouse uterus and fetus undergoes major shifts during pregnancy.     

Age-related augmentation of phosphorylase b kinase in hepatic tissue from the glycogen-storage-disease (gsd/gsd) rat.

The effects of food deprivation on body weight, liver weight, hepatic glycogen content, glycogenolytic enzymes and blood metabolites were compared in young and old phosphorylase b kinase-deficient (gsd/gsd) rats. Although the concentration of glycogen in liver from 9-week-old female gsd/gsd rats (730 mumol of glucose equivalents/g wet wt.) was increased by 7-8% during starvation, total hepatic glycogen was decreased by 12% after 24 h without food. In 12-month-old male gsd/gsd rats the concentration of liver glycogen (585 mumol of glucose equiv./g wet wt.) was decreased by 16% and total hepatic glycogen by nearly 40% after food deprivation for 24 h. Phosphorylase b kinase and phosphorylase a were present at approx. 10% of the control activities in 9-week-old gsd/gsd rats, but both enzyme activities were increased more than 3-fold in 12-month-old affected rodents. It is concluded that the age-related ability to mobilize hepatic glycogen appears to result from the augmentation of phosphorylase b kinase during maturation of the gsd/gsd rat.     

A flow-dialysis method for obtaining relative measures of association constants in calmodulin-metal-ion systems.

A flow-dialysis apparatus suitable for the study of high-affinity metal-binding proteins has been utilized to study calmodulin-metal exchange as a measure of relative calmodulin-metal association constants. Calmodulin labelled with radioactive 153Gd was dialysed against buffer containing various competing metal ions. The rate of label exchange was monitored by a gamma-ray scintillation detector. Competing metals used were Ca2+ and Cd2+, and the lanthanides Gd3+, Eu3+, La3+ and Lu3+. All exchange processes were first-order, and two categories of metal were found: Ca2+ and Cd2+ in one, the lanthanides comprising the other. In addition calmodulin-metal complexes with radioactive 109Cd and 45Ca released the bound label without any competing metal being added to the buffer. The kinetics of this metal loss can be described by two consecutive first-order processes, and the fraction of label associated with each rate can be determined. Studies of phosphodiesterase activation by calmodulin show Cd2+ and calmodulin to cause 80% of the maximum activation found when Ca2+ and calmodulin are used.     

Eucaryotic chromosome transfer: production of a murine-specific cosmid library from a neor-linked fragment of murine chromosome 17.

We recently developed a procedure for the molecular analysis of specific mammalian chromosomal fragments. This procedure allows for the transfer of contiguous chromosomal fragments, varying in size from a fraction to several centimorgans in length, from the donor cell of one species into a recipient cell of a different species. Specifically, we inserted a neor gene, encoded by a recombinant retrovirus, into the murine major histocompatibility complex (MHC). Metaphase chromosome transfers with this neor-tagged chromosome into recipient hamster, primate, and canine fibroblasts produced a panel of primary neor transferents, each containing a portion of, or all of, the murine MHC. A cosmid library was made from one such transferent, CHMD(D)B1. Cosmid clones were divided, using species-specific repeat probes, into those containing murine (donor) DNA sequences and those containing sequences derived from the recipient cell. The murine-specific cosmids were clustered into overlapping DNA segments by restriction enzyme digest analysis of the cosmid DNAs coupled with Southern blot analysis with, as probes, murine-specific repeat sequences and nick-translated murine genomic DNA. These cosmid clusters were analyzed for their position within or outside of the MHC, using recombinant mouse strains, and for the presence within them of known murine MHC genes.