A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural. DNA preparations relative to controls.     

Biodiversity of sweetpotato (iPomoea batatas,Convolvulaceae) in Southern Mexico

Between 1987 and 1992 the phytogeographic region of southern Mexico was explored during three collecting trips made in search of cultivated and wild germplasm of the sweetpotato (Ipomoea batatas). The first trip was made in 1987, when we collected wild species in Ipomoea section Batatas found in the southeastern and southwestern regions of Mexico. A second trip was made in 1990, when we collected accessions of the cultivated species as well as wild species in the southeast, southwest and northeast. The third and final trip was oriented at identification, characterization and collecting seeds in the ecological niches ofI.tabascana andI.umbraticola. As a result of the three trips we collected 165 accessions of cultivated and wild germplasm with populations dispersed in 147 localities in 15 states of the Mexican region: Veracruz, Tabasco, Campeche, Chiapas, Oaxaca, Yucatán, Guerrero, Michoacán, San Luis Potosí, Hidalgo, Querétaro, Tamaulipas, Guanajuato, Puebla and México. Of the total accessions some 64 (38.3%) were of the cultivated species including nine accessions of feral material, and 103 accessions (61.7%) were of wild species made up of 59 accessions of seven species in the section Batatas, 37 of other species in the family Convolvulaceae, and seven yet to be determined. We have identified the largest genetic biodiversity in six localities of five states: Tabasco, Oaxaca, Michoacán, San Luis Potosí, and Puebla. Biodiversity maintenance in this region is associated with the day-of-the-dead festivities.     

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.     

Comparative evolutionary rates of introns and exons in murine rodents

Analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous genes of mouse and rat was used to compare patterns of evolutionary change between introns and exons. The mean of the absolute difference in length (measured in base pairs) between the two species was nearly five times as high in the case of introns as in the case of exons. The average rate of nucleotide substitution in introns was very similar to the rate of synonymous substitution in exons, and both were about three times the rate of substitution at nonsynonymous sites in exons. G+C content of introns and exons of the same gene were correlated; but mean G+C content at the third positions of exons was significantly higher than that of introns or positions 1–2 of exons from the same gene. G+C content was conserved over evolutionary time, as indicated by strong correlations between mouse and rat; but the change in G+C content was greatest at position 3 of exons, intermediate in introns, and lowest at positions 1–2 in introns. Received: 23 December 1996 / Accepted: 1 April 1997     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Hyperthermia,antipyretics and function of polymorphonuclear leukocytes.

Whether hyperthermia (temperature, 40 degrees C), salicylates, acetaminophen or phenacetin has an adverse effect on polymorphonuclear leukocyte (PMNL) function was examined. Migration experiemnts were carried out in Boyden chambers with bacterial chemotactic factor as the attract, and bactericidal assays were done with Staphylococcus aureus and serum from an AB blood group donor as a source of opsonins. PMNL viability was determined by the trypan blue exclusion method. Neither hyperthermia nor any of the drugs tested affected PMNL viability adversely, but sodium salicylate and phenacetin suppressed PMNL migration. Early staphylococcal killing was greater at 40 degrees C; however, after 2 hours the converse was true. Bactericidal activity was suppressed by acetylsalicylic acid, sodium salicylate and phenacetin. Hence it appears PMNL function is similar at 37 degrees and 40 degrees C but that some commonly used antipyretics have an adverse effect on PMNL activity.     

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.     

IN VIVO RNA SYNTHESIS WITHIN THE RAT NODOSE GANGLIA

Abstract— The in vivo synthesis of RNA in the rat nodose ganglia has been studied following the intravenous introduction of ortho-[³²P]phosphate. Analysis of the labelled RNA upon 2.2% polyacrylamide gels was performed. Rapidly-labelled heterodisperse RNA. with a size range of 10S-30S was detected after 3 h exposure to the isotope. Two peaks, of size 28S and 12S-14S. were also observed. The former is thought to be processed 28S rRNA whereas the latter is consistent with its designation as 'message-like' RNA (mlRNA). A rapidly turning-over phosphorylated non-nucleic acid contaminant prevented clear interpretation of the 4S region of the gel at short labelling times. However, this material was not present when the exposure time was increased to 24 h. At this time, only the stable RNA species 28S and 18S rRNA and 4S tRNA, were detected.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.