Isolation and partial characterization of DNA polymerases from Crithidia fasciculata.

Two types of DNA polymerase activity were partially purified from Crithidia fasciculata. The alpha-type, DNA polymerase A, was of high molecular weight and sensitive to N-ethylmaleimide, whereas the beta-type, DNA polymerase B, was of low molecular weight and resistant to N-ethylmaleimide. Phosphocellulose chromatography revealed multiple peaks of DNA polymerase A activity the properties of which, such as pH optimum, salt sensitivity, utilization of synthetic template-initiator complexes and response to DNA polymerase inhibitors were similar. The response of the C. fasciculata DNA polymerase A enzymes to some of these inhibitors and utilization of poly(rA) X oligo(dT)11 showed these enzymes to be markedly different from mammalian DNA polymerase alpha.

Changes in size and shape of chromatin particles after successive removal of histones.

The preparations of whole chromatin, chromatin selectively depleted of histone f1, depleted of all lysine-rich histones (f1, f2b, f2a2), and DNA was studied by viscosimetric and light scattering methods. The obtained results were used for calculation of the dimensions and packing ratios of DNA for the preparations studied. The packing ratio in whole chromatin is 7.2 and is almost unaffected by selective removal of histone f1 (6.9), but decreases on successive removal of the remaining four histones, the decrease being dependent more on the quantity than the kind of the dissociated histones.

On the arrangement of histones in chromatin.

It was found that nucleoprotein particles formed after DNase I action on calf thymus chromatin contain single-stranded DNA fragments, associated with histones only by ionic linkages. These results suggest that histones in chromatin are bound ionically only to one polynucleotide strand of double-helical DNA, protecting it against nucleolytic attack.

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode, Heterodera glycines.

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.

Blood parameters as consistent predictors of nestling performance in great tits (Parus major) in the wild.

Fourteen blood profile variables were analysed in 12-day-old nestlings of great tits (Parus major) in the wild. Except for plateletocrit and platelet distribution width, the traits showed a consistent pattern of variation, with significant intra-brood repeatabilities; they were shown to be significant predictors of nestling performance as measured by survival from hatching to fledging. It is concluded that all blood characteristics that show consistent within-brood variation may be useful as indicators of some aspects of nestling condition/health status in wild birds.

End structure and mechanism of packaging of bacteriophage T4 DNA.

We analyzed by restriction enzyme digestion the end structure of T4 phage DNA by comparing mature, concatemeric, first-packaged, and incompletely packaged DNAs. The structure of mature DNA was also studied using 3' end labeling with terminal transferase. Our data support the hypothesis that T4 DNA packaging is not initiated at specific packaging initiation sequences on the concatemeric precursor (cos or pac site mechanisms) but by a different packaging mechanism.

An endonuclease activity of chicken erythrocyte nuclei and mononucleosomes.

Endogenous nuclease is present in the nuclear sap of chicken erythrocyte nuclei. This enzyme resembles the nuclease of mammalian nuclei in requirements for bivalent cations and in production of large chromatin fragments that gradually decrease in size, but differs in that the products do not go through the stage of discrete bands on gel electrophoresis. Endogenous nuclease and micrococcal nuclease are also detectable in mononucleosomes prepared from chicken erythrocytes with the aid of micrococcal nuclease. Both nucleases are extractable with 0.35 M NaCl, and both are inhibited by pTp. In the absence of Ca2+, the micrococcal nuclease is totally inactive, whereas the endogenous nuclease shows a low level of activity.

Study of barley endonucleases and alpha-amylase genes.

We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes. This enzyme(s) has been designated as a "linker"-specific nuclease(s). This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells. The linker-specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin. The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone. Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue. Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin. In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha-amylase cDNA clone. In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid. This is the first report of the presence of a linker-specific nuclease activity in plant cells.

Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family.

A 34,000-Da protein (P34) is one of the four major soybean oil body proteins observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated organic solvent-extracted oil bodies from mature seeds. P34 is processed during seedling growth to a 32,000-Da polypeptide (P32) by the removal of an amino-terminal decapeptide (Herman, E.M., Melroy, D.L., and Buckhout, T.J. (1990) Plant Physiol, in press). A soybean lambda ZAP II cDNA library constructed from RNA isolated from midmaturation seeds was screened with monoclonal antibodies directed against two different epitopes of P34. The isolated cDNA clone encoding P34 contains 1,350 base pairs terminating in a poly(A)+ tail and an open reading frame 1,137 base pairs in length. The open reading frame includes a deduced amino acid sequence which matches 23 of 25 amino-terminal amino acids determined by automated Edman degradation of P34 and P32. The cDNA predicts a mature protein of 257 amino acids and of 28,641 Da. The open reading frame extends 5' from the known amino terminus of P34 encoding a possible precursor and signal sequence segments with a combined additional 122 amino acids. Prepro-P34 is deduced to be a polypeptide of 42,714 Da, indicating that the cDNA clone apparently encodes a polypeptide of 379 amino acids. A comparison of the nucleotide and deduced amino acid sequences in the GenBank Data Bank with the sequence of P34 has shown considerable sequence similarity to the thiol proteases of the papain family. Southern blot analysis of genomic DNA indicated that the P34 gene has a low copy number.

Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs.

We have characterized two cDNA clones for 24 kDa soybean oleosin, the seed oil body membrane protein. Differences in the predicted amino acid sequences of the two clones and the presence of a doublet on immunoblots indicate that 24 kDa oleosin exists in at least two isoforms in soybean. The predicted amino acid sequence also contains a unique carboxy terminal region that is dominated by a series of different tandem amino acid repeats.

A soybean vacuolar protein (P34) related to thiol proteases is synthesized as a glycoprotein precursor during seed maturation.

We have examined the synthesis, posttranslational processing, and localization of soybean P34, a member of the papain superfamily. P34 has been identified as a constituent of oil storage organelles or oil bodies isolated from seed lysates and has been assumed to be one of the oil body proteins. Electron microscopic immunocytochemistry with a monoclonal antibody demonstrated that P34 is localized in the protein storage vacuoles but not in the oil bodies. Immunocytochemical observations of partially disrupted seed cells showed that the association of P34 with oil bodies appears to occur as a consequence of cell lysis. In vitro synthesis of P34 results in the formation of a 46-kDa polypeptide that increases to 47 kDa due to core glycosylation by canine microsomes. In vivo synthesis studies in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation, indicate that pro-P34 is 47 kDa. Since the cDNA sequence of prepro-P34 contains a single putative glycosylation site in the precursor domain, we conclude that P34, like a few other vacuolar proteins, is synthesized as a glycoprotein precursor. Pulse-chase experiments showed that the processing of pro-P34 to mature P34 occurs in a single step and that this posttranslational cleavage occurs on the carboxyl side of an Asn, which is typical of seed vacuolar proteins. Pro-P34 (47 kDa) is detected in immunoblots of maturing seeds. Analysis of RNA indicates that the P34 genes are expressed only during seed maturation and that the P34 mRNA is related to other thiol protease mRNAs detectable in other organs and plants. Unlike other seed thiol proteases that are synthesized only after seed germination, P34 accumulates during seed maturation.

Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.

Binding-protein expression is subject to temporal, developmental and stress-induced regulation in terminally differentiated soybean organs.

Binding protein (BiP) is a widely distributed and highly conserved endoplasmic-reticulum luminal protein that has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides. Analysis of cDNA sequences and genomic blots indicates that soybeans (Glycine max L. Merr.) possess a small gene family encoding BiP. The deduced sequence of BiP is very similar to that of other plant BiPs. We have examined the expression of BiP in several different terminally differentiated soybean organs including leaves, pods and seed cotyledons. Expression of BiP mRNA increases during leaf expansion while levels of BiP protein decrease. Leaf BiP mRNA is subject to temporal control, exhibiting a large difference in expression in a few hours between dusk and night. The expression of BiP mRNA varies in direct correlation with accumulation of seed storage proteins. The hybridization suggests that maturing-seed BiP is likely to be a different isoform from vegetative BiPs. Levels of BiP protein in maturing seeds vary with BiP mRNA. High levels of BiP mRNA are detected after 3 d of seedling growth. Little change in either BiP mRNA or protein levels was detected in maturing soybean pods, although BiP-protein levels decrease in fully mature pods. Persistent wounding of leaves by whiteflies induces massive overexpression of BiP mRNA while only slightly increasing BiP-protein levels. In contrast single-event puncture wounding only slightly induces additional BiP expression above the temporal variations. These observations indicate that BiP is not constitutively expressed in terminally differentiated plant organs. Expression of BiP is highest during the developmental stages of leaves, pods and seeds when their constituent cells are producing seed or vegetative storage proteins, and appears to be subject to complex regulation, including developmental, temporal and wounding.