Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.      

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Pure Culture Studies of Erwinia carotovora with 3,5-Diiodo-4-Hydroxybenzonitrile

Interactions of ioxynil (3,5-diiodo-4-hydroxybenzonitrile) with a pure culture of Erwinia carotovora grown in a glucose-simple salts medium were studied. Growth of E. carotovora was inhibited by ioxynil and, to a lesser extent, by its acid form at 25 and 50 mug/ml. Growth was not inhibited by the amide or ester forms of ioxynil or p-hydroxybenzonitrile at the same concentrations. E. carotovora could be trained to grow in 50 mug or higher concentrations of ioxynil per ml by serial transfers of the organism through increasing ioxynil concentrations. No degradation or detoxification of ioxynil was detected. Toxicity tests indicated that, in the adapted culture, cell-free supernatant fluid remained toxic to a nonadapted culture. Adaptation of E. carotovora resulted in a lengthened lag phase, a decreased growth rate, and very few adverse effects on the total population. The adapted resistant culture retained this characteristic only when ioxynil was present. Adaptation was demonstrated to be a physiological variation, not a selection of a mutant or of preexisting resistant cells. Ioxynil slightly stimulated the respiration rate of E. carotovora and moderately inhibited that of an adapted culture. Because the respiration rate of an adapted culture in the absence of ioxynil surpassed that of a parent culture still in the presence of ioxynil, a competition of two alternate routes of electron transport is implied. These data support the conclusion that an alternate growth mechanism is involved in the adaptation mechanism.     

Targeted ablation of pituitary gonadotropes in transgenic mice.

LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.     

Shoot regeneration from petunia leaf discs as a function of explant size,configuration and benzyladenine exposure

The effect of explant size, configuration, and duration of benzyladenine exposure on shoot organogenesis in Petunia x hybrida Ultra Salmon leaf discs was determined. Leaf discs of 13, 10, 8, 5, and 3 mm diameter, and 10 mm with a 5 o4 3 mm hole and 13 mm with an 8 or 5 mm hole were tested. The smallest size and configuration for maximum shoot production and consistency of regeneration was a 10 mm whole disc. This was followed by the 8, 10 with a 3 mm hole, 5, 10 with a 5 mm hole and 3 mm disc respectively. Of all configurations tested, the 13 mm disc and the 13 mm disc with a 5 mm hole produced the most shoots. Exposure of leaf discs to benzyladenine-containing medium for 4 days or less resulted in no shoots being formed. Exposure for 10 days or more induced shoot regeneration on all discs tested. A delay of more than 2 days after excision, before plating on benzyladenine-containing medium, decreased shoot regeneration significantly. The explant configurations tested showed that: the area to cut edge ratio was a good predictor of shoot number for either of the two configurations when considered separately; and there are factors other than area to cut edge ratio involved in relating shoot production to explant configuration and size.Abbreviation BA benzyladenine     

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Analysis of Bacterial Spatial Patterns at the Initial Stage of Biofilm Formation

Using sophisticated microscopy techniques, we observed the spatial pattern of bacteria colonizing a sterile 316L stainless steel coupon as bulk water containing bacteria flowed across the coupon. The experiments used stainless steel of differing roughness and surface chemistry. The ultimate goal was to identify surface features which influence bacterial adsorption. The immediate statistical goal was to distinguish patterns consistent with complete spatial randomness from patterns showing regularity or aggregation. This goal was accomplished by using modified analyses of distance functions commonly applied in field ecology. The method protected against a potential multiple comparisons problem. For the null value of the distance function, we calculated tolerance envelopes such that the tolerance level was simultaneous for all distances of concern. Computer simulation experiments showed that the nominal level was accurate. The methodology was effective for detecting and describing patterns of colonization known not to be completely spatially random.