Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1

The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.     

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

A DNA polymerase from the archaeon Sulfolobus solfataricus shows sequence similarity to family B DNA polymerases.

The gene encoding the thermostable DNA polymerase from the archaeon Sulfolobus solfataricus (strain MT 4) was isolated by means of two degenerate oligonucleotide probes. They were designed on the basis of partial enzyme amino acid sequences. The gene was found to encode a 882 residues polypeptide chain with a deduced molecular mass of about 100 kDa. By comparison with other archaeal genes, putative regulatory sites were identified in the gene-flanking regions. By computer-assisted homology search, several sequence similarities among S. solfataricus and family B DNA polymerases were found. In addition, conserved sequence motifs, implicated in the 3'-5' exonuclease activity of E. coli DNA polymerase I and shared by various family A and B DNA polymerases, were also identified. This result suggests that the proofreading domains of all these enzymes are evolutionarily related.     

Small sequence insertions within the branch point region dictate alternative sites of lariat formation in a yeast intron.

The problem of intron recognition in S. cerevisiae appears to be in part solved by the strong conservation of intron encoded splicing signals, in particular the 5' GUAUGU and the branch point UACUAAC which interact via base pairing with the RNA components of U1 and U2 snRNPs respectively. Nevertheless, the mere presence of such signals is insufficient for splicing to occur. In the S. cerevisiae ACT1 intron, a silent UACUAAC-like sequence (UACUAAG) is located 7 nucleotides upstream of the canonical branch point signal. In order to investigate whether other factors, in addition to the U2-UACUAAC base-pair interactions, affect branch point selection in yeast, we created a cis-competition assay by converting the UACUAAG to a strong branch point signal (UACUAAC). If simply having a canonical UACUAAC sequence were sufficient for lariat formation, a 1:1 ratio in usage of the two signals should have been observed. In this double branch point intron, however, the downstream UACUAAC is utilized preferentially (4:1). Results obtained from the analyses of numerous sequence variants flanking the two UACUAAC sequences, demonstrate that non-conserved sequences in the branch point region are able to define lariat formation. Consequently, we conclude that U2 base-pairing is not the only requirement determining branch point selection in yeast, and local structure in the vicinity of the branch point could play a critical role in its recognition.     

Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium

Ammonium sulfate, a typical component of crystallization media of proteins, stabilizes an inactive conformation of pig muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, in the presence of ammonium sulfate the reconstitution of the catalytically active holoenzyme from the apoenzyme and NAD is not instantaneous, as in the case of enzymes from Bacillus stearothermophilus and the Mediterranean lobster Palinurus vulgaris. With pig muscle enzyme, at pH 6.0, the time course of formation of the characteristic Racker band can be monitored by a rapid mixing stopped flow technique. Activation follows a single exponential curve with a rate constant independent of the concentration of both NAD and protein and, therefore, appears to be limited by a slow protein isomerization (k = 7 +/- 2 s-1). Accordingly, when the apoenzyme is simultaneously exposed to NAD and either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate, the ensuing reactions (the redox and the acylation steps, respectively) are kinetically limited by the same protein isomerization. At pH 7.0 and 8.0, however, two among the four active sites react with NAD at an unmeasurably high rate, while the other two sites behave as they do at pH 6.0. When the pig muscle apoenzyme is preincubated and allowed to react with either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate before the rapid mixing with NAD, both the redox reaction and the NAD-dependent activation of apo-acyl-enzyme toward arsenolysis become unmeasurably fast. Similarly, when the sulfate in the medium is replaced by ions such as phosphate and citrate, the reconstitution of the active holoenzyme is practically instantaneous. Thus, the slow protein isomerization observed in the presence of sulfate and abolished by competing substrates and anions is diagnostic of a structural state of the pig muscle apoenzyme, which is induced by sulfate ions bound within the enzyme active site.