Species-specific patterns of DNA bending and sequence.

Nucleotide sequences in the GenEMBL database were analyzed using strategies designed to reveal species-specific patterns of DNA bending and DNA sequence. The results uncovered striking species-dependent patterns of bending with more variations among individual organisms than between prokaryotes and eukaryotes. The frequency of bent sites in sequences from different bacteria was related to genomic A + T content and this relationship was confirmed by electrophoretic analysis of genomic DNA. However, base composition was not an accurate predictor for DNA bending in eukaryotes. Sequences from C. elegans exhibited the highest frequency of bent sites in the database and the RNA polymerase II locus from the nematode was the most bent gene in GenEMBL. Bent DNA extended throughout most introns and gene flanking segments from C.elegans while exon regions lacked A-tract bending characteristics. Independent evidence for the strong bending character of this genome was provided by electrophoretic studies which revealed that a large number of the fragments from C.elegans DNA exhibited anomalous gel mobilities when compared to genomic fragments from over 20 other organisms. The prevalence of bent sites in this genome enabled us to detect selectively C.elegans sequences in a computer search of the database using as probes C.elegans introns, bending elements, and a 20 nucleotide consensus sequence for bent DNA. This approach was also used to provide additional examples of species-specific sequence patterns in eukaryotes where it was shown that (A) greater than or equal to 10 and (A.T) greater than or equal to 5 tracts are prevalent throughout the untranslated DNA of D.discodium and P.falciparum, respectively. These results provide new insight into the organization of eukaryotic DNA because they show that species-specific patterns of simple sequences are found in introns and in other untranslated regions of the genome.     

Specificity of DNA uptake in genetic transformation of gonococci

Genetic transformation of gonococci to streptomycin resistance was inhibited by homologous DNA or by DNA from related $\text{Neisseriae}$, but not by high concentrations of heterologous DNAs. Gonococci were capable of adsorbing large quantities (up to about 50 μg per 10⁸ cells) of both homologous and heterologous DNA, which could not be eluted by strong shearing forces. Treatment with externally added DNase removed virtually all the heterologous DNA while a small fraction of the homologous DNA, not influenced by the presence of excess heterologous DNA, remained cell-bound in a form resistant to nuclease treatment. Competing homologous DNA suppressed nuclease-resistant binding. These findings suggest that gonococci have two types of DNA binding components at their surface. Competence of gonococci for genetic transformation undergoes a rapid decay if the cells are incubated with homologous (but $\text{not}$ with heterologous) DNA.     

Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis.

Simian Cos-1 cells were transfected electrically with the plasmid pCH110 carrying the beta-galactosidase gene. The efficiency of transfection was determined by a transient expression of this gene. When the plasmid was introduced into a cell suspension 2 s after pulse application, the transfection efficiency was shown to be less than 1% as compared with a prepulse addition of DNA. Addition of DNAase to suspension immediately after a pulse did not decrease transfection efficiency, thus the time of DNA translocation was estimated to be less than 3 s. The use of electric treatment medium, in which the postpulse colloid-osmotic cell swelling was prevented, did not affect the transfection efficiency. These results contradict both assumptions of free DNA diffusion into cell through the long-lived pores and of involvement of osmotic effects in DNA translocation. Transfection of cells in monolayer on a porous film allowed creation of the spatial asymmetry of cell-plasmid interaction along the direction of electric field applied. A pulse with a polarity inducing DNA electrophoresis toward the cells resulted in the 10-fold excess of transfection efficiency compared with a pulse with reverse polarity. Ficoll (10%) which increases medium viscosity or Mg2+ ions (10 mM) which decrease the effective charge of DNA, both reduced transfection efficiency 2-3-fold. These results prove a significant role of DNA electrophoresis in the phenomenon considered. The permeability of cell membranes for an indifferent dye was shown to increase noticeably if the cells were pulsed in the presence of DNA. This indicates a possible interaction of DNA translocated with the pores in an electric field, that results in pore expansion.     

Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.     

A 26-base-pair repetitive sequence specific for Neisseria gonorrhoeae and Neisseria meningitidis genomic DNA.

Two-dimensional heteroduplex mapping of Neisseria gonorrhoeae genomic DNA revealed a number of spots, indicating the existence of repetitive sequences. When one of the spots was extracted and used as a probe for Southern blot analysis, two HindIII bands (11.0 and 3.6 kilobases [kb]) of the genomic digest hybridized with approximately equal intensity. The 3.6-kb fragment was cloned and found to contain two different types of repeated sequence. One type was approximately 1.1 kb in length and was found at least twice in the entire genome. The other consisted of a 26-base-pair family GT(C/A)C(Py)G(Pu)TTTTTGTTAAT(Py)C(Pu)CTATA (Py, pyrimidine; Pu, purine) that was repeated at least 20 times in the entire genome. This repetitive sequence was found also in Neisseria meningitidis but not in various other gram-negative bacteria.     

Species-specific detection of Listeria monocytogenes by DNA amplification.

The polymerase chain reaction was used to detect and specifically identify Listeria monocytogenes. A 174-bp region of the listeriolysin O gene was shown to be specifically amplified in L. monocytogenes but not in other species of Listeria or in a number of other gram-positive and gram-negative organisms. Less than 50 organisms could routinely be detected by a procedure involving two rounds of 35 amplification cycles each and without the need for subsequent hybridization with labeled probes.     

Species-specific cell adhesion of enteropathogenic Escherichia coli is mediated by type IV bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhoea in humans. Localized adherence of EPEC onto intestinal mucosa was reproduced in an in vitro adherence assay with cultured human epithelial cells. We found that the efficiency of EPEC adherence to a mouse-derived colonic epithelial cell line, CMT-93, was remarkably lower than its adherence to human-derived intestinal cell lines, such as Intestine-407 or Caco-2. Although EPEC did adhere to some cell lines derived from non-human species, fixing the cells with formalin to inactivate one or more formalin-sensitive factors allowed us to observe species-specific differences in EPEC adherence. In contrast to these results, an EPEC mutant that is defective in bundle-forming pili (BFP) production adhered as efficiently to CMT-93 cells as to Caco-2 cells. Furthermore, Citrobacter rodentium expressing BFP adhered to Caco-2 cells much more efficiently than to CMT-93 cells. Finally, a purified BfpA-His6 fusion protein showed higher affinity for Caco-2 cells than for CMT-93 cells, and inhibited EPEC adherence. Following BFP-mediated adherence, secretion of EspB from adherent bacteria and reorganization of F-actin in the host cells was observed. EPEC adhering to CMT-93 cells induced far less secretion of EspB, or reorganization of F-actin in the host CMT-93 cells, than did EPEC adhering to Caco-2 cells. These results indicated that BFP plays an important role in the cell-type-dependent adherence of EPEC and in the progression to the later steps in EPEC adherence.     

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.     

Delayed-type hypersensitivity is mediated by a sequence of two different T cell activities

Classical 24- to -48 hr delayed-type hypersensitivity (DTH) skin reactions are preceded by an early skin swelling reaction that peaks 2 hr after challenge. The ability to elicit this early component of DTH is T cell dependent and is also dependent on tissue mast cells and release of serotonin, mainly from these mast cells. The current study presents pharmacologic and kinetic evidence that the late response depends on the occurrence of the early response. A variety of pharmacological agents known to deplete, prevent release of, or block the activity of serotonin, when given just before skin challenge, blocked both the early and late components of DTH, but had no effect when given (even repeatedly) after the occurrence of the early component. Thus, the serotonin-dependence of the 24-hr component of DTH represents a dependence on the early component in which serotonin release is required. A temporal dependence of the late component of DTH on the early component was also demonstrated. The early and late phases occur at different times in recipients of sensitized T cells, depending on the interval between transfer and challenge, but there is a fixed 10- to 12-hr gap. Delayed onset of the late component occurs in recipients challenged immediately after transfer and appears to be due to a delay in the onset of the early component. This delay can be abolished by adoptive cell transfer into mice that are able to elicit a normal early component because of prior transfer of T cells that are able to mediate just an early component. On the basis of these findings, we conclude that DTH consists of a cascade of events. T cells mediating the early aspect of DTH release antigen-specific factors that, upon encountering antigen activate local serotonin-containing cells, such as mast cells, to release serotonin, which opens gaps between adjacent endothelial cells. Through these interendothelial gaps a second T cell population enters the extravascular space and interacts with local antigen to induce the late response by releasing the chemoattractant lymphokines that are classically associated with DTH and that cause recruitment of bone marrow-derived circulating leukocytes to infiltrate the reaction site. The ability of the second T cell population to mediate the late component of DTH is independent of further release of serotonin.     

Human cell DNA replication is mediated by a discrete multiprotein complex

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.     

Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.     

Vacuolar malate uptake is mediated by an anion-selective inward rectifier

Electrophysiological studies using the patch‐clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current–voltage relations of the tonoplast were dominated by a strongly inward‐rectifying anion‐selective channel that was active at cytoplasmic‐side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage‐dependent gating of a 3‐pS malate‐selective ion channel, which showed a half‐maximal open probability at ?43 mV. Also, the time‐averaged unitary currents following a step to a negative voltage corresponded to the time‐dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion‐selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent K_m of 2.5 mm malate^2? activity), a selectivity sequence of fumarate^2? > malate^2? > Cl^? > maleate^2– ≈ citrate^3–, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 µm niflumate reversibly blocked the activity of the 3‐pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 µm^?2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3‐pS, inward‐rectifying, anion‐selective channel represents the principal pathway for malate influx into the vacuole.