Comparative studies of Babesia spp. from white-tailed and sika deer

Babesia odocoilei from white-tailed deer (Odocoileus virginianus) in Texas (USA) and B. capreoli isolated from sika deer (Cervus nippon) in Ireland were compared morphologically and antigenically. Babesia odocoilei and B. capreoli paired pyriforms resembled each other closely when in sika deer, but B. odocoilei pyriforms in white-tailed deer were slightly different. Babesia odocoilei in white-tailed deer also differed from B. odocoilei and B. capreoli in sika deer in the frequency of its developmental forms. Indirect immunofluorescence antibody test titres showed that there was some antigen cross-reactivity, but not as much as between B. capreoli and the bovine parasite, B. divergens. The Babesia spp. from deer that we studied appear to be distinct but related species. The low infectivity of B. odocoilei for a splenectomised sika deer suggests that sika deer in North America are probably not very susceptible to this parasite in the wild.     

Effect of N-alkyl substituents on the DNA binding properties of meso-tetrakis (4-N-alkylpyridinium-4-yl)porphyrins and their nickel derivatives

Resonance Raman, NMR, and visible spectroscopies, as well as viscosity and equilibrium dialysis studies were used to assess the effect of the N-alkyl substituent of meso-tetrakis(4-N-alkylpyridinium-4-yl)porphyrin cations on DNA binding. The DNAs studied include the native DNA, calf thymus DNA (CT DNA), the synthetic polynucleotides [poly(dGdC)]2 and [poly(dAdT)]2, and the oligonucleotide d(TATACGTATA)2. Both the porphyrins and the metalloporphyrins containing Ni(II) were examined with the N-alkyl = propyl (TPrpyP(4) and NiTPrpyP(4)) and 2-hydroxyethyl (TEtOHpyP(4) and NiTEtOHpyP(4)). The results were compared to those from the parent porphyrins with the N-methyl substituent (TMpyP(4) and NiTMpyP(4)). For almost all the comparisons made, the new porphyrin cations gave results very similar to those for the TMpyP(4) species. The resonance Raman study indicated that for the three DNA polymers all the Ni species were in the four-coordinate form when bound to all three polymers. It is suggested that both TPrpyP(4) and TEtOHpyP(4) bind to GC regions of DNA in the same intercalative manner as TMpyP(4) with the N-alkyl substituent extended into the solvent. For AT regions of DNA, the binding of TPrpyP(4) and TEtOHpyP(4) is nonintercalative, as found previously for TMpyP(4). The NiPrpy(4) and NiTEtOHpyP(4) cations bind to these polymers in a similar manner to the apo-porphyrins. The similar Raman spectral changes for the three Ni porphyrins upon addition of [poly(dAdT)]2 suggest that partial intercalation is not occurring because models indicate that it would be difficult to accommodate the bulkier N-alkyl substituents.     

In vitro micropropagation and plant establishment of muscadine grape cultivars (Vitis rotundifolia)

Shoot apical meristems were used to establish regenerative axillary bud cultures of 9 muscadine grape cultivars. Meristems taken from 10 cm long shoots had less contamination (3%) and a higher survival rate (94%) than those from shorter or longer shoots. Of media tested, MS, 1/2 MS, and C₂D resulted in equivalent shoot proliferation rates, whereas, WPM produced stunted shoots. When pooling results for 3 cultivars, 5, 10 and 20 M BA and 5 M TDZ produced the highest average number of shoots per cultured apex (3.4–3.8). However, shoots produced with TDZ were stunted and did not root well. For rooting of shoots directly in potting mix, a rooting powder pretreatment significantly increased the number of roots per shoot but did not affect percent rooting or root length. For rooting in vitro, 1 M NAA significantly increased all parameters measured. Although more shoots rooted in vitro than in vivo (77% vs. 46%), the latter was judged preferable since acclimatized plants were produced in less time and a major culture step was eliminated. Significant differences among cultivars were noted for measured responses in all experiments.Abbreviations BA benzyladenine - Kin kinetin - MS Murashige & Skoog (medium) - NAA naphthaleneacetic acid - TDZ thidiazuron - WPM woody plant medium     

Vacuum UV CD spectra of homopolymer duplexes and triplexes containing A.T or A.U base pairs.

Vacuum UV circular dichroism (CD) spectra were measured down to 174 nm for five homopolymers, five duplexes, and four triplexes containing adenine, uracil, and thymine. Near 190 nm, the CD bands of poly[d(A)] and poly[r(A)] were larger than the CD bands of the polypyrimidines, poly[d(T)], poly[d(U)], and poly[r(U)]. Little change was observed in the 190 nm region upon formation of the duplexes (poly[d(A).d(T)], poly[d(A).d(U)], poly[r(A).d(T)], poly[r(A).d(U)], and poly[r(A).r(U)]) or upon formation of two of the triplexes (poly[d(T).d(A).d(T)] and poly[d(U).d(A).d(U)]). This showed that the purine strand had the same or a similar structure in these duplexes and triplexes as when free in solution. Both A.U and A.T base pairing induced positive bands at 177 and 202 nm. For three triplexes containing poly[d(A)], the formation of a triplex from a duplex and a free pyrimidine strand induced a negative band centered between 210 and 215 nm. The induction of a band between 210 and 215 nm indicated that these triplexes had aspects of the A conformation.     

Application of flow karyotyping in prenatal detection of chromosome aberrations

This paper describes the application of bivariate flow karyotyping to (1) classification of chromosomes isolated from cultures of cells taken by amniocentesis and (2) detection of numerical and structural aberrations. Chromosomes were isolated from primary cultures 2-5 wk after amniocentesis, stained with Hoechst 33258 and chromomycin A3, and analyzed using dual beam flow cytometry. Information about chromosome DNA content and DNA base composition was derived from the locations of the peaks in the flow karyotypes, each peak being produced by one or more chromosome types with similar DNA content and DNA base composition. Information about the relative frequency of each chromosome type was determined on the basis of the relative volume of the peak for that chromosome type. Cytogenetic information determined on the basis of flow karyotypes was compared with that obtained by visual analysis following G-banding. Variability among the peak means and volumes in flow karyotypes was determined from analyses of 50 normal amniocyte cultures. Numerical aberrations involving chromosomes 21, 18, and Y were detected correctly in all of 28 analyses, including eight in a blind study. Structural aberrations involving chromosomes 1, 2, 3, 6, 9-12, 13, 14, 15, 21, and 22 were detected in all of seven cultures in a blind study. Flow karyotypes proved to be insensitive to small, normally occurring chromosome polymorphisms detected by banding analysis. In addition, a few samples were erroneously scored as having numerical aberrations.     

N-terminal amino acid sequence analysis of the subunits of pea photosystem I

Six 'core' subunits of pea photosystem I have been isolated and their N-terminal amino acid sequences determined by gas-phase or solid-phase sequencing. On average more than thirty residues were determined from the N-terminus of each polypeptide. This sequence analysis has revealed three polypeptides with charged N-terminal regions (21, 17 and 11 kDa subunits), one polypeptide with a predominantly hydrophobic N-terminal region (9 kDa subunit), one polypeptide which is cysteine-rich (8 kDa subunit) and one which is alanine-rich (13 kDa subunit).     

Effects of Chromosomal Inversion on Cell Fitness in Escherichia Coli K-12

In an effort to learn what factors might mitigate the establishment of Escherichia coli variants bearing major chromosomal rearrangements, we have examined the effects on cell growth of two inversions between rRNA operons. One of these inversions, IN(rrnD-rrnE), had been propagated in a commonly used subline of E. coli K-12 for approximately 30 yr before its discovery, a fact that illustrates the absence of obvious detrimental effects associated with the inversion. We found that culturing under conditions requiring repeated transition from stationary phase to rapid growth led to the replacement of IN(rrnD-rrnE) cells by cells that had undergone either of two types of additional chromosomal inversion: one type fully restored the wild-type order, while the other partially restored it. The partial reinversion was also between rrn operons, but it left a small transposition. The tendency for overgrowth by these revertants persisted through several rounds of periodic selection. In contrast, the other inversion, IN(rrnG-rrnE), was associated with severe, detrimental effects. The effects of IN(rrnG-rrnE) were also alleviated by full or partial reinversion. The probable relationship between the severity of the effects caused by the inversions and the degree of displacement of the replication origin is discussed. Spontaneous inversion events between rrn operons separated by 18% of the chromosome were estimated to occur at a frequency of roughly 10(-5). If extended to natural situations, the growth disadvantage together with the relatively high frequency of reinversion suggest that clones of cells with an inversion between these rrn operons would be readily overgrown by revertants.     

Circular intermediates with missing nucleotides in the conversion of supercoiled or nicked circular to linear duplex DNA catalyzed by two species of BAL 31 nuclease

The extracellular nucleases from Alteromonas espejiana BAL 31 can catalyze the endonucleolytic and/or exonucleolytic hydrolysis of duplex DNA in response to a variety of alterations, either covalent or noncovalent, in DNA structure. The nuclease can exist as at least two kinetically and molecularly distinct protein species. The two species that have been studied, called the 'fast' (F) and 'slow' (S) nucleases, both readily convert negatively supercoiled DNAs to linear duplex molecules and accomplish this conversion through the formation of a circular duplex intermediate containing usually a single interruption in one strand. It is further shown that most of these intermediates contain gaps arising from the removal in a processive manner of one or more nucleotide residues after the introduction of the initial strand break (nick). Considering only the intermediates with gaps, the average number of missing residues is 6.3 +/- 0.5 and 2.8 +/- 0.3, respectively, for DNA acted upon by the F and S enzymes independently of the extent of conversion of supercoiled DNA. The nicks and gaps are bounded by 3'-hydroxyl and 5'-phosphoryl termini. When singly nicked circular DNA is used as the substrate, conversion to the linear duplex form occurs predominantly through a gapped circular intermediate with the same average numbers, within experimental error, of missing nucleotides for the respective nuclease species as found when supercoiled DNA is the substrate. The conversion to linear duplex DNA is much slower when nicked circular DNA is the substrate compared to that found when supercoiled DNA is the starting material.     

Antigenic variation among group A streptococcal M proteins. Nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins

The 1479-base pair (bp) nucleotide sequence of the serotype 5 M protein gene (smp5) from Streptococcus pyogenes contains three distinct types of tandemly repeated sequences, designated A, B, and C. Repeat A (21 bp x 6, in the 5'-half of smp5), shares no homology with the types 6 or 24 M protein genes (Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1986) J. Biol. Chem. 261, 1677-1686; Mouw, A. R., Beachey, E. H., and Burdett, V. (1988) J. Bacteriol., in press). Repeat B (75 bp x 3.6, in the center of smp5) is also present in the M6, but not in the M24 gene. Repeat C (105 bp x 2.7, just distal to the B repeats) shares homology with repeats in both the M6 and M24 genes. All three genes share extensive homology in their 3'-halves and in 5' sequences encoding the N-terminal signal peptides, but between these two regions there are highly variable sequences that are responsible for antigenic diversity. These relationships suggest that both intergenic and intragenic recombination has occurred during the evolution of distinct M protein serotypes. All three M proteins contain conserved hydrophobic and proline-rich sequences at their C-terminal ends, suggestive of a membrane anchor and a peptidoglycan spanning region.