The acceleration of linear DNA during pulsed-field gel electrophoresis

The velocity and orientation of T4 and lambda DNA have been measured for the first 20 s during pulsed-field gel electrophoresis in order to clarify the DNA motions that occur. For a square pulse with field strength E = 10 V/cm, the velocity of lambda DNA increases gradually to 10.5 microns/s in 1.0 s, declines to 8.6 microns/s, and then rises to a plateau value of 9.3 microns/s after 4 s. T4 DNA behaves similarly, but more slowly. Parallel measurements of fluorescence-detected linear dichroism show that the DNA becomes substantially aligned with its chain axis parallel to the electrophoretic field E after the pulse is applied. The alignment also shows an overshoot, an undershoot, and a plateau comparable to those seen for velocity. When the field strength increases, both the velocity and the alignment reach their peaks more quickly. For all field strengths and both molecular weights, the velocity peak occurs when the molecular center of mass has moved 0.3 to 0.5 L, where L is the chain contour length. A qualitative model is provided.     

Characterization and taxonomic status of tick spiroplasmas: a review

Three serologically distinct groups of spiroplasmas have been recovered from ticks. Spiroplasma mirum strains (from rabbit ticks, Haemaphysalis leporispalustris) and Y32 group (VI) spiroplasmas (from Ixodes pacificus) are the only spiroplasmas to have a clear association with these arthropods. Group (VI) spiroplasmas are distinguished by an unusual nonhelical morphology and their capacity to hemadsorb guinea pig erythrocytes. S. mirum strains are unique in their ability to induce cataracts or lethal brain infections in a number of young vertebrates and in their virulence for the chick embryo. The 277F spiroplasma, while initially recovered from a pool of rabbit ticks (H. leporispalustris), is related by certain serological and genetic properties to spiroplasmas in the S. citri complex (serogroup I). These relationships suggest that the 277F spiroplasma may not be a natural inhabitant of the rabbit tick.     

Improved Cultivation Systems for Isolation of the Colorado Potato Beetle Spiroplasma

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.     

Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.      

Direct isolation in cell-free medium of a spiroplasma fromHaemaphysalis leporispalustris (Acari: Ixodidae) in Maryland

A spiroplasma isolate, was obtained from rabbit ticks (Haemaphysalis leporispalustris) taken from cottontail rabbits in Maryland by inoculation of tick suspensions into SP-4 medium. The isolate was indistinguishable from an experimental vertebrate pathogen (suckling mouse cataract agent spiroplasma) when tested with other plant and tick spiroplasmas in growth inhibition, deformation, and metabolism inhibition tests. The isolated organism had a pathogenic profile for suckling rats and embryonated chicken eggs that differed significantly from that of other suckling mouse cataract agent strains. This is the first report of a direct spiroplasma isolation from ticks in cell-free medium, and confirms the specific association of spiroplasmas of the suckling mouse cataract agent serogroup with rabbit ticks.     

Lampyridae (Coleoptera): A plethora of mollicute associations

Beetles (Coleoptera) harbor many species ofAcholeplasma andSpiroplasma (division Tenericutes, class Mollicutes). Mollicutes were isolated from guts and/or hemocoels of firefly beetles (Lampyridae) from the United States (Maryland and West Virginia), Ecuador, and Tobago. Firefly beetles were frequent hosts for the group XIV spiroplasma, isolated from Ellychnia corrusca, and the group XIX spiroplasma, isolated fromPhoturis spp. The most unusual feature of the firefly-mollicute association is the carriage of four Mycoplasma species. Recent phylogenetic studies indicate that these species are members of a clade that includes a vertebrate pathogen,Mycoplasma mycoides. The high rate of occurrence ofMycoplasma species (which are, otherwise, infrequent in insects) in lampyrid beetles suggests that the association is significant. The unusual light-producing physiology of lampyrids (which is dependent on large pools of energy) and the production of large amounts of cardenolides from cholesterol (a critical growth factor for many mollicutes) may favor colonization by mollicutes. Offprint requests to: K. J. Hackett.     

Wall-less prokaryotes from fall flowers in central United States and Maryland

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland old fields represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.