Molecular cloning of the F8 fimbrial antigen from Escherichia coli

Abstract The genetic determinant coding for the P-specific F8 fimbriae was cloned from the chromosome of the Escherichia coli wild-type strain 2980 (O18:K5:H5:F1C, F8). The F8 determinant was further subcloned into the Pst I site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned counterpart was demonstrated. The cloned F8 fimbriae and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepithelial cells. The cloned F8 determinant was well expressed in a variety of host strains.     

A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis

A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis.     

Analysis of a repetitive DNA family of two closely related chironomids,Chironomus thummi thummi andCh. thummi piger (Diptera) by density-gradient centrifugation,melting analysis and restriction analysis

The DNAs of the two subspecies ofChironomus thummi, Ch. th. thummi andCh. th. piger, were investigated by CsCl density-gradient centrifugation, melting analysis and restriction analysis including Southern hybridization with AT-rich, highly repetitive DNA sequences. The melting analysis of density-fractionatedCh. th. thummi andCh. th. piger DNA has shown that thethummi DNA contains an early melting DNA fraction, which is enriched in the light fractions of the density gradient. The DNA fraction is also present inpiger DNA though in lower concentration. Restriction and Southern analysis of density fractionatedthummi andpiger DNA has revealed that there are two tandemly-repetitive DNA-sequence families that hybridize with this AT-rich, early melting DNA fraction. One sequence is characterized by anHae-III site and a basic repeat length of 130 ± 15 bp and the other by aCla-1 restriction site and a basic repeat length of 120 ± 4 bp. These sequences are present in much higher concentrations in the genome ofCh. th. thummi when compared toCh. th. piger, and are hence correlated to the higher DNA content of theCh. th. thummi genome.     

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.     

Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light-dependent increase in [³H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [³H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light-enhanced incorporation of [³H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [³H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus-enhanced turnover of PI in some tissues, this light-enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (²²Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of ²²Na in light but not in dark.     

Ultrastructure of trypan blue induced ocular defects: I. Retina and lens

The histological and cytological basis of trypan blue-induced ocular defects were studied using scanning and transmission electron microscopy. Microphthalmic and anophthalmic eyes of 16-day rat fetuses were utilized from dams exposed to a teratogenic dose of trypan blue. Retinal and lenticular anlagen were specifically examined for architectural and cellular changes. Nearly all severely abnormal eyes showed no evidence of retina development: Of 41 such eyes, only two retinal rudiments were observed. Those eyes with mild microphthalmia always demonstrated retinae although architectural changes were present. In every abnormal eye, some degree of lenticular morphogenesis was always present. Lenses were small, displaced in the eye field, and arrested at the lens vesicle stage. Lens cells were markedly undifferentiated and thus lacked most of the cytological features normally present at this developmental stage. Neither retinal nor lenticular rudiments were necrotic despite major architectural and cytological disturbances. The data offer three conclusions: First, the absence of necrosis suggests that trypan blue causes developmental arrest in this eye model; second, absence of retinae is most likely due to primary failure of optic vesicle development; third, lack of lens differentiation is attributed to absence of the retina, the primary lens inducer.     

A Novel abscisic acid metabolite from cell suspension cultures of Nigella damascena

The structure of a novel abscisic acid metabolite isolated from cell suspension cultures of Nigella damascena fed [2-¹⁴C]abscisic acid was iden     

Light- and cytidine-dependent phosphatidylinositol synthesis in photoreceptor cells of the rat

Incorporation of [3H]inositol into phosphatidylinositol (Pl) in isolated rat retinas is enhanced by light and by the addition of cytidine to the incubation media. In retinas preincubated with [3H]inositol in dark, [3H]inositol was chased into Pl in light by addition of unlabeled cytidine and was chased out of Pl in light by addition of unlabeled cytidine plus inositol. Autoradiograms of retinas show a heavy density of silver grains over photoreceptor cell inner segments (with chase-in) and a loss of labeling (with chase-out). Exogenous cytidine and inositol were shown to enhance not only the turnover of Pl within photoreceptor cells but the synthesis of Pl as well; in media supplemented with these precursors, approximately 50% of [14C]glycerol and 25% of [32Pi]incorporated into lipid in light were associated with Pl. These results suggest that availability of both cytidine and inositol may play a role in the light-dependent changes in Pl metabolism within photoreceptor cells.     

Identification of Tn2401, a transposon encoding multiresistance to aminoglycosides

A transposable element, Tn2401, was found in a clinical isolate of Pseudomonas aeruginosa. Tn2401 had a size of 7190 nucleotides and encoded aminoglycoside 3'-phosphotransferase and aminoglycoside 6'-N-acetyltransferase. The sequence encoding the former enzyme was homologous with that of Tn903. Pseudomonas aeruginosa strains harbouring this transposon were resistant to kanamycin, neomycin, lividomycin, ribostamycin, paromomycin, netilmycin, tobramycin, dibekacin, gentamicin, sisomicin, and butirosin.