Regulation of nuclear membrane assembly and maintenance during in vitro maturation of mouse oocytes: role of pyruvate and protein synthesis

Summary In the absence of a suitable energy source, mouse oocytes cultured in vitro resume, but fail to complete, meiotic maturation. However, little is known about the underlying mechanisms leading to this meiotic failure. We utilized pyruvate-deficient medium to test for the role of pyruvate throughout the meiotic maturation process. Germinal vesicle-stage (GV) oocytes underwent germinal vesicle breakdown (GVBD), but failed to form a polar body when cultured continuously in pyruvate-free medium. However, when GV oocytes were preincubated for 4 h in pyruvate-free medium containing dibutyryl cyclic adenosine monophosphate (dbcAMP) and then cultured in pyruvate-free medium, GVBD was markedly inhibited. Preincubation of GV oocytes in dbcAMP and cycloheximide, followed by culture in cycloheximide only, also inhibited GVBD. A longer preincubation period was required in the cycloheximide-dbcAMP case (12 h) than in pyruvate-free-dbcAMP medium situation (4 h). Strikingly, reassembly of the nuclear membrane without polar body formation was observed following GVBD in oocytes continuously cultured in pyruvate-free medium. The reassembled nuclear membrane increased in size with continued culture, and it surrounded partially-decondensed chromatin. Nuclear membrane reassembly also occurred in oocytes which had undergone GVBD during continuous culture in medium containing only cycloheximide. Reformation of nuclear membranes after GVBD was confirmed by electron-microscopic analyses of oocytes cultured in pyruvate-free medium or in the presence of cycloheximide. We conclude that both pyruvate and protein synthesis are required for nuclear membrane disassembly, whereas lack of pyruvate or protein synthesis is associated with interruption of the metaphase state and reassembly of the nuclear membrane. The evidence suggests that assembly and maintenance of an intact nucleus and its disintegration are all amenable to regulation by pyruvate, possibly via mechanism(s) involving protein synthesis.     

Polymorphism in a ferritin H gene from chromosome 6p

Summary This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.     

Genomic characterization ofCampylobacter jejuni by field inversion gel electrophoresis

The genome ofCampylobacter jejuni was characterized by field inversion gel electrophoresis (FIGE) after digestion with three rare-cutting restriction endonucleases. The restriction enzymesSac II (5-CCGCGG),Sal I (5-GTCGAC), andSma I (5-CCCGGG) were found to produce 13, 5, and 8 fragments respectively from theC. jejuni genome. The fragment sizes ranged from 1.6 kb to 1300 kb, which gaveC. jejuni a genome size of approximately 1900 kb. Furthermore, thegly A and rRNA genes ofC. jejuni were localized to specific fragments by use of Southern analysis, and thegly A gene was shown to be closely linked to one of the three rRNA genes.     

Phylogenetic and evolutionary implications of nuclear ribosomal DNA variation in dwarf dandelions (Krigia,Lactuceae, Asteraceae)

Restriction site and length variations of nrDNA were examined for 51 populations of seven species ofKrigia. The nrDNA repeat ranged in size from 8.7 to 9.6 kilobase (kb). The transcribed region, including the two ITSs, was 5.35 kb long in all examinedKrigia populations. In contrast, the size of the nontranscribed IGS varied from 3.35 to 4.25 kb. Eight different types of length-variations were identified among the 51 populations, including distinct nrDNA lengths in the tetraploid and diploid populations of bothK. biflora andK. virginica. However, a few variations were detected among populations of the same species or within a cytotype. All populations ofKrigia sect.Cymbia share a 600 bp insertion in IGS near the 18 S gene, and this feature suggests monophyly of the section. AllKrigia spp. had a conjugated type of subrepeat composed of approximately 75 basepairs (bp) and 125 bp. Base modifications in the gene coding regions were highly conserved among species. Forty-five restriction sites from 15 enzymes were mapped, 24 of which were variable among populations. Only four of the variable sites occurred in the rRNA coding region while 20 variable sites were detected in the noncoding regions. Collectively, 25 enzymes generated about 66 restriction sites in each nrDNA; this amounts to about 4.3% of the nrDNA repeat. A total of 50 restriction sites was variable, 28 of which were phylogenetically informative. Phylogenetic analyses of site mutations indicated that two sections ofKrigia, sect.Cymbia and sect.Krigia, are monophyletic. In addition, relationships among several species were congruent with other sources of data, such as cpDNA restriction site variation and morphology. Both length and restriction site variation supported an allopolyploid origin of the hexaploidK. montana. The average sequence divergence value inKrigia nrDNA was 40 times greater than that of the chloroplast DNA. The rapid evolution of nrDNA sequences was primarily due to changes of the IGS sequences.     

Nucleotide and primary sequence of a major rice prolamine

A recombinant cDNA clone encoding a major rice seed storage prolamine was isolated by antibody screening of a cDNA lambda gt 11 library. This clone contained a single open reading frame encoding a putative rice prolamine precursor (Mr 17,300). In contrast to other cereal prolamines, the primary sequence of the rice prolamine was devoid of any major tandem repetitive sequences, a feature prevalent in all cereal prolamines studied to date. No significant homology was detected between the rice prolamine and other cereal prolamines, indicating that the rice gene evolved from unique ancestral DNA segments.     

ACCURACY OF PHYLOGENETIC-ESTIMATION METHODS UNDER UNEQUAL EVOLUTIONARY RATES

A comparative study of the accuracy of three different approaches to phylogenetic estimation was made on simulated data with differing rates of change in different lineages. The three approaches were maximum likelihood, maximum parsimony, and phenetic clustering. The data were generated by simulating genetic drift with different population sizes over a simple four-species tree topology. Although the accuracy of all three approaches was found to be dependent on the number of loci (characters), maximum likelihood was found to perform considerably and consistently better than maximum parsimony or phenetic clustering.     

Site in the cat-86 regulatory leader that permits amicetin to induce expression of the gene.

Expression of the plasmid gene cat-86 is induced in Bacillus subtilis by two antibiotics, chloramphenicol and the nucleoside antibiotic amicetin. We proposed that induction by either drug causes the destabilization of a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the cat coding sequence. The destabilization event frees the ribosome-binding site, permitting the initiation of translation of cat-86 mRNA. cat-86 induction is due to the stalling of a ribosome in a leader region of cat-86 mRNA, which is located 5' to the RNA stem-loop structure. A stalled ribosome that is active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To test the hypothesis that a leader site 5' to codon 6 permits a ribosome to stall in the presence of an inducing antibiotic, we inserted an extra codon between leader codons 5 and 6. This insertion blocked induction, which was then restored by the deletion of leader codon 6. Thus, induction seems to require the maintenance of a precise spatial relationship between an upstream leader site(s) and leader codon 6. Mutations in the ribosome-binding site for the cat-86 leader, RBS-2, which decreased its strength of binding to 16S rRNA, prevented induction. In contrast, mutations that significantly altered the sequence of RBS-2 but increased its strength of binding to 16S rRNA did not block induction by either chloramphenicol or amicetin. We therefore suspected that the proposed leader site that permitted drug-mediated stalling was located between RBS-2 and leader codon 6. This region of the cat-86 leader contains an eight-nucleotide sequence (conserved region I) that is largely conserved among all known cat leaders. The codon immediately 5' to conserved region I differs, however, between amicetin-inducible and amicetin-noninducible cat genes. In amicetin-inducible cat genes such as cat-86, the codon 5' to conserved region I is a valine codon, GTG. The same codon in amicetin-noninducible cat genes is a lysine codon, either AAA or AAG. When the GTG codon immediately 5' to conserved region I in cat-86 was changed to AAA, amicetin was no longer active in cat-86 induction, but chloramphenicol induction was unaffected by the mutation. The potential role of the GTG codon in amicetin induction is discussed.