Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.     

Isolation and Characterization of a Photosystem II Core Complex Depleted in the 43 kDa-Chlorophyll-Binding Subunit

The photosystem II core complex purified from digitonin extractsof spinach chloroplasts was resolved into two chlorophyll-proteincomplexes by digitonin polyacrylamide gel electrophoresis aftertreatment with 1 M potassium thiocyanate. One of the chlorophyll-proteincomplexes resolved consisted of 47, 32, 30 and 9 kDa polypeptidesand the other was complementally composed of only the 43 kDapolypeptide. The former complex was highly active in the photoreductionof 2, 6-dichlorophenol indophenol by 1,5-diphenylcarbazide andretained all of the components responsible for the electrontransport from the secondary electron donor (Z) to the primaryelectron acceptor (Q_A). EPR signal II_fast and II_slow were alsopreserved in this complex although their hyperfine structureswere largely modified. The complex was estimated to contain1.8 molecules of plastoquinone A as well as 1.5, 3.7 and 3.9molecules of cytochrome b559, pheophytin and ß-carotene,respectively, per Q_A. These results indicate that potassiumthiocyanate specifically removes the 43 kDa polypeptide fromthe PS II core complex leaving the electron transport systemin an almost intact state. (Received June 17, 1987; Accepted October 23, 1987)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)     

Detection of fimbriae and fimbrial antigens on the oral anaerobe Bacteroides gingivalis by negative staining and serological methods

We screened 63 clinical isolates of Bacteroides gingivalis from eight different laboratories for the presence of fimbriae by negative staining and by immunological methods. Techniques used were bacterial agglutination, Ouchterlony immunodiffusion and Western immunoblotting analysis using rabbit anti-fimbriae and anti-fimbrilin sera raised against fimbriae and fimbrilin (a constituent protein of B. gingivalis fimbriae) from B. gingivalis strain 381. In 49 of the 51 strains tested, fimbriae were clearly detected by negative staining, and 30 (60%) of the fimbriate strains were positive in all three of the immunological assays. A total of 37 strains (75%) were positive by immunoblotting analysis, which was the most reliable of the serological methods used in this study. The study shows that the majority of B. gingivalis strains are fimbriate, and that these fimbriae are immunologically related to the fimbriae of B. gingivalis strain 381. Molecular heterogeneity of fimbrilin was discovered by the immunoblotting analysis, when different strains were compared. With most of the strains, including strain 381, the antifimbrilin serum reacted with a protein of apparent molecular mass 43 kDa, but with 15 strains the immuno-reactive protein had an apparent molecular mass of 46 kDa.     

Annual changes in the testicular activity of the river sculpin, Cottus hangiongensis Mori, with emphasis on the occurrence of aberrant spermatids during spermatogenesis

Annual changes in the spermatogenetic activity of the testis were studied histologically in the river sculpin. Coitus hangiongensis , sampled monthly from a river in southern Hokkaido, Japan. A pair of sperm reservoirs, consisting of many anastomosing lacunae, was present along the dorsomedian edge of the paired testes, and functioned also as a sperm-transporting system instead of the typical sperm duct. Spermatogenesis occurred actively in August, yielding an increasing number of mature spermatozoa in October. This process advanced, but slowly during the succeeding winter months, until March. The testis became functionally mature during the spawning period in April and May. In July, small numbers of spermatocytes were found to have appeared already, which indicated a relatively short period of post-spawning testicular regression. In November, germinal cysts containing aberrant binuclear spermatids began to appear within the seminal lobules. The paired nuclei of aberrant spermatids gradually enlarged, and the cells were released into the lumina of the seminal lobules simultaneously with the release of mature spermatozoa from the germinal cysts. During the functional maturity stage, lumina of seminal lobules which had expelled mature spermatozoa to sperm reservoirs became filled with these abnormal bodies. Discussion includes the occurrence of aberrant spermatids which resulted in the formation of 'spermatid masses' as has been described in other cottids.     

Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Morphology and ontogeny of stem xylem elements inSarcandra glabra (Thunb.) Nakai (Chloranthaceae): Additional evidence for the occurrence of vessels

Very recentlySarcandra, which had long been known as the only vesselless genus in Chloranthaceae, was found by Carlquist to have vessels in root secondary xylem. The present study further shows on the basis of observations of the xylem ontogeny that vessels occur in stem metaxylem ofSarcandra glabra as well, thus offering additional evidence for the occurrence of vessels in the genus, virtually in all Chloranthaceae. Metaxylem elements of the stem are thicker than the other tracheary elements in general and have scalariform pittings at the end wall, and their ontogeny indicates that, as the surrounding cytoplasm disintegrates, pit membranes at the end wall disappear at least in some elements, resulting in a perforated end wall, i.e., vessel perforation. The present study further shows thatChloranthus spicatus, which is closely related toSarcandra, may have an incomplete perforation plate because of retaining membranes at places on the plate. An evolutionary state of the “vesselless” condition in Chloranthaceae is discussed.