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.     

Monoclonal antibodies reveal evolutionary conservation of alternative splicing of the alpha A-crystallin primary transcript

Because of their specificity and sensitivity, monoclonal antibodies are powerful tools in studies of protein structure and function. Therefore, we raised monoclonal antibodies against alpha A-crystallin and identified the antigenic determinant for two of these antibodies. Applying limited-digestion methods, we show that the region spanning residues 158-168 of alpha A-crystallin contains the epitope for the two monoclonal antibodies. These monoclonals were then used to study the occurrence in the lenses of different vertebrates of the elongated alpha Ains-crystallin chain, a product of alternative splicing. It appears that the mutational event resulting in the alternative splicing pattern of the alpha A-crystallin gene took place at least 70 million years ago. This alternative splicing phenomenon has been maintained in rodents and some other, unrelated mammals, but disappeared again in most mammalian lineages.     

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.     

Affinity purification of human deoxycytidine kinase: avoidance of structural and kinetic artifacts arising from limited proteolysis

Homogeneous deoxycytidine kinase has been isolated from leukemic human T-lymphoblasts by affinity chromatography based on a multisubstrate analog, deoxycytidine 5'-adenosine 5"'-P1,P4-tetraphosphate (dCp4A). Chromatography of extract treated with protease inhibitors yielded a monomeric polypeptide, inasmuch as the Mr of the native protein, 59,300, is comparable to the value of 52,000 from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric pH was 6.1. But, enzyme isolated without protease inhibitors exhibited two fragments of Mr = 30,000 and 33,000, suggesting that proteolytic cleavage of the parental polypeptide had occurred during affinity chromatography. Both the parental and proteolyzed enzymes phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. However, the proteolyzed enzyme had an increased apparent Km for deoxycytidine. In consequence of this, a mixture of the two forms produced bimodal kinetic plots, whereas linear kinetics were displayed by each form alone.     

Membrane-associated NAD+ glycohydrolase from rabbit erythrocytes is solubilized by phosphatidylinositol-specific phospholipase C

NAD+ glycohydrolase (NADase) present on the surface of rabbit erythrocytes is a membrane-bound ectoenzyme that can be solubilized by phosphatidylinositol-specific phospholipase C (PIPLC). As much as 70% of the cell-associated NADase was made soluble by treatment with PIPLC. The portion of NADase that remained cell-associated after an initial PIPLC treatment proved to be resistant to subsequent solubilization attempts. Further analysis showed that release of NADase from erythrocytes could not be attributed to the action of proteinases or phospholipase C. Erythrocytes obtained from other mammals were analyzed and found to have variable amounts of PIPLC-susceptible NADase. Practically, this finding can be used to easily solubilize membrane-bound NADase as a first step in its purification.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.     

Digitalis-like pregnanes. Cardiac and renal effects of a glycoside of 14 beta-hydroxyprogesterone

The synthesis of the glucoside, 3 beta-[(beta-D-glucopyranosyl)oxy]-14-hydroxy-14 beta-pregn-4-en-20-one, a 14 beta-hydroxyprogesterone glucoside (14 beta-OHP-glu), is described. This compound has an IC50 of 1 microM in a [3H]ouabain binding assay, and is about 10 times more potent than the aglycone. Like 14 beta-hydroxyprogesterone, the glucoside enhances contractility of isolated cardiac muscle. 14 beta-OHP-glu or ouabain, when infused at comparable doses into the renal artery of the anesthetized rat, markedly increases urine volume. Whereas ouabain significantly enhances urinary potassium excretion with little or no effect on sodium excretion, 14 beta-OHP-glu promotes a marked natriuresis with no significant effect on potassium excretion.