Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Characterization of yeast ribosomal DNA fragments generated by EcoR1 restriction endonuclease

Summary The action of Escherichia coli restriction endonuclease R1 (EcoR1) on DNA isolated from Saccharomyces cerevisiae (strain MAR-33) generates three predominent homogenously sized DNA fragments (species of 1.8, 2.2 and 2.5 kilo nucleotide base pairs (KB). Many DNA species of molecular weight greater than 2 million daltons can be recognized upon incomplete EcoR1 digestion of yeast DNA. Four additional DNA species ranging from 0.3–0.9 KB can be identified as the second major class of EcoR1-yeast DNA products.Hybridization with radioactive ribosomal RNA (rRNA) and competition with nonradioactive rRNA show that of the three predominent EcoR1-yeast DNA species, the 2.5 KB species hybridizes only with the 25S rRNA while the lighter 1.8 KB species hybridizes with the 18S rRNA. The intermediate DNA species of 2.2 KB hybridizes to a small extent with the 25S rRNA and could be a result of the presence of the 2.5 KB DNA species. The mass proportions and hybridization values of these 3 DNA species account for about 60% of the total ribosomal DNA (rDNA).The 5EcoR1-yeast DNA species of less than 0.9 KB (4 major and 1 minor species) hybridize to varying degrees with the 2 rRNA and can be grouped in two classes. In one class there are 3 DNA species that hybridize exclusively with the 18S rRNA. In the second class there are 2 DNA species that besides hybridizing predominently with the 25S rRNA also hybridize with the 18S rRNA. The 7 EcoR1-yeast DNA species (excluding the 2.2 KB DNA species) that hybridize with the two rRNA account for nearly a 5 million dalton DNA segment, which is very close to the anticipated gene size of rRNA precursor molecule. If the 2.2 KB DNA species is a part of the rDNA that is not transcribed or 5 sRNA then the cistron encoding the rRNA in S. cerevisiae has at least 8 EcoR1 recognition sites resulting in 8 DNA fragments upon digestion with the EcoR1. Consideration is given to the relationship of the rRNA species generated by EcoR1 digestion and the chromosomes containing ribosomal cistrons.     

Effects of temperature,metabolic and cytoskeletal inhibitors on the rate of BHK cell adhesion to polystyrene

Adhesion of baby hamster kidney fibroblasts (BHK cells) to a Falcon tissue culture flask was measured under various physiological conditions. While 75–80% of the fibroblasts adhere at temperatures from 19–50°, cellular adhesion decreased dramatically below 19°. Less than 10% of the cells adhere to the substratum even after prolonged incubations at temperatures of 8° or below. This lack of adhesion at low temperatures cannot be overcome by the application of increased gravitational force to the cells. No correlation exists between cellular ATP concentrations or respiration rates and the rate of cell adhesion to the substratum. One millimolar Na F and 1 mM 2,4 dinitrophenol together lower cellular ATP concentration by 95% but adhesion is reduced by only 50%. NaN₃ and KCN greatly lower cellular ATP concentrations without a corresponding inhibition of adhesion. Inhibition of cellular respiration by these compounds occurs at lower concentrations than does the inhibition of adhesion. Two micrograms/milliliters of cytochalasin B inhibits adhesion by 90%, 0.1 mM vinblastine sulphate or colchicine by less than 50% and 50 μg/ml colcemid by less than 30%. Fixing the cells with formaldehyde, hardening their membranes with ZnCl₂ or treating the cells with toluene, all cause an inhibition in adhesion. Again, application of increased gravitational force cannot overcome these latter inhibitions of BHK cell adhesion to the surface of the flasks.     

Restriction analysis of tandemly repeated yeast ribosomal RNA genes

Summary Ribosomal RNA (rRNA) genes of Saccharomyces cerevisiae are clustered in a DNA repeat unit of 5.9 megadaltons with the gene order 5S-18S-5.8S-25S rRNA (Nath and Bollon, 1977). By using two restriction endonucleases, EcoRII and HindII, which generate DNA fragments that span contiguous portions of two repeat units, we report that the rRNA gene clusters are tandemly repeated without the intervention of additional spacer DNA.The treatment of yeast DNA with the restriction endonucleases EcoRII and HindII result in the generation of 4 different DNA fragments that are of varying sizes and which hybridize with rRNA. The largest DNA fragments, 3.30 megadaltons in the case of HindII and 3.67 megadaltons in the case of EcoRII, encompass regions that code for the two opposite end regions of the 35S precuursor-rRNA. These two end regions are joined by a constant DNA segment of about 0.9 megadaltons in size of which a 0.08 megadalton segment codes for 5S rRNA. Since the 35S precursor-rRNA includes the 5.8S, 18S and 25S rRNA most of the repeat units containing the 4 rRNA coding genes in yeast are linked to each other contiguously without any intervening spacer DNA.A composite map of the DNA restriction fragments obtained by the action of the restriction endonucleases EcoRI, EcoRII, HindII and HindIII on the 5.9 megadalton repeat unit is presented. Some striking features concerning the location of the restriction sites are noted. Of the total 17 DNA restriction sites present on each repeat unit, 9 are located at or near the 3 transcribed spacer regions contained in the 5 megadalton DNA segment that codes for the 35S precursor-rRNA. The 3 transcribed spacer regions in the 35S precursor-rRNA include the two external transcribed spacer regions and an internal transcribed spacer region, the latter representing the 5.8S rRNA.