Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo.

The U1 snRNP is known to play a critical role in spliceosome assembly, at least in part through base pairing of its RNA moiety to the substrate, but many details remain to be elucidated. To further dissect U1 snRNA function, we have analyzed 14 single point mutations in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast Schizosaccharomyces pombe. Three of the four alleles previously found to support growth of Saccharomyces cerevisiae are lethal in S. pombe, implying a more critical role for the 5' end of U1 in fission yeast. Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site. The importance of U1 function in S. pombe is further underscored by the lethality of several single point mutants not examined previously in S. cerevisiae. In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth. Each viable mutant was tested for impaired splicing of three different S. pombe introns. Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor. Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site. Although point mutations at the 5' end of U1 have no discernible effect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment. Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast.     

Relationship between elevated lipid peroxides,vitamin E deficiency and hypertension in preeclampsia

Preeclampsia or pregnancy-induced hypertension is a major cause of both maternal and fetal-neonatal morbidity and mortality. The deficiency of vitamin E can cause accumulation of lipid peroxidation products, which, in turn, can induce vasoconstriction. This study has examined any evidence of increased cellular lipid peroxidation and accumulation of malonydialdehyde (MDA, an end product of lipid peroxidation) in pregnancy-induced hypertension and any relationship between the elevated MDA and lower vitamin E levels with hypertension in pregnant women. EDTA-Blood was collected from pregnant women at the time of delivery. Plasma vitamin E was determined by HPLC; MDA by the thiobarbituric acid-reactivity. Subjects with diastolic blood pressure(DBP) 90 mm Hg were considered hypertensive (HT) and with <90 mm Hg normotensive (NT). Data (Mean±SE) from 49 NT and 11 HT women show that HT has significantly lower vitamin E (22±1 vs 27±1 nmole/ml, p<0.03) and elevated MDA levels (0.56±0.06 vs 0.43±0.02 nmole/ml, p<0.03) compared to NT; the ages and gestational ages of women were similar. Among all women, there was a significant positive relationship between DBP and MDA levels (r=0.27, p<0.05), and a significant negative relationship between vitamin E levels and DBP (–0.36, p<0.005), and a significant negative relationship between MDA and vitamin E levels (r=–0.27, p<0.05). Thus, HT women's plasma has significantly lower E and higher MDA levels, and DBP significantly correlates with the extent of vitamin E deficiency and increased MDA levels. This study suggests a relationship between elevated lipid peroxidation and lower vitamin E levels and hypertension in pregnancy (preeclampsia).     

Improved techniques for use of the triploid cell marker in the axolotl, Ambystoma mexicanum

Techniques for using the triploid cell marker for studying cell lineage during the development and regeneration of the axolotl limb are described. Triploid animals possess cells with three nucleoli while diploid animals possess cells with two nucleoli. We have developed a technique for isolating the limb dermis as a sheet of cells for whole-mount analysis of cellular ploidy. Whole-mount tissue preparations as well as paraffin-embedded sectioned tissues were stained specifically for nucleoli with bismuth. Cell counts from a number of triploid and diploid dermal preparations show that (1) diploid dermal cells never possess three nucleoli, (2) the frequency of trinucleolate cells in whole-mount triploid dermal preparations is not 100% but varies between animals from 30 to 76%, (3) within a single triploid animal, the frequency of trinucleolate cells in different dermal preparations is constant. These data establish the usefulness of this technique and emphasize the need for appropriate control cell counts when using the triploid cell marker in the axolotl.     

The three-dimensional structure of theCyphomandra betacea chloroplast peripheral reticulum

Summary The peripheral reticulum (PR) inCyphomandra betacea chloroplasts originates as vesicles budding from the inner membrane of the chloroplast envelope which elongate to form tubules then aggregate or branch to form discrete PR units. Individual PR units of many coiled tubules may be connected with other units by narrow tubules. Serial sectioning revealed the discrete units to be approximately 650–1,000 nm wide, 400–500 nm high and 500–600 nm deep and to possess a compact morphology. TheCyphomandra PR structure is compared to the morphologies of chloroplast reticula reported for other plant species. A scheme to group PR from different species into 3 distinct morphological categories is outlined and discussed.     

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Artificial insemination of subtropical commercial beef cattle following synchronization with cloprostenol (ICI 80996): I. Fertility

Two trials were conducted over a two-year period with 519 cycling Bos taurus x Bos indicus heifers and cows. The objectives of these trials were: 1) To compare fertility of artificial insemination at the cloprostenol-induced estrus and the naturally occurring estrus, 2) To evaluate the fertility of artificial insemination at a predetermined time (Timed AI) following an estrous synchronization regime with cloprostenol (CLP) and 3) To define the optimum interval from a second CLP treatment for Timed AI. In Trial I, 128 animals were assigned to four treatments: 1) Controls, which were inseminated at the natural occurring estrus; 2) timed AI at 72 hr and again at 96 hr post-second CLP; 3) Timed AI at 72 hr post-second CLP and 4) AI at the CLP-induced estrus. Trial II included 391 heifers distributed among six treatments; 1) Timed AI between 70 and 90 hr post-second CLP; 2) Sham AI between 70 and 90 hr post-second CLP, 3) Chute Stress between 70 and 90 hr post-second CLP; 4) AI at the CLP-induced estrus; 5) Control-AI at the naturally occurring estrus and 6) Non-treated and exposed to fertile bulls. The fertility of the animals artificially inseminated at the CLP-induced estrus was similar to that of insemination at the naturally occurring estrus in Trial I and Trial II (30 vs 46%; 37 vs 38%, respectively). The first service pregnancy rates of the animals bred at a predetermined time were similar to those bred at the CLP-induced estrus in Trial I, but lower in Trial II (P < .01).     

Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small

The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA. We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs. DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene. Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich. The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.     

On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase.

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.