Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Modification of radiation damage in the canine kidney by hyperthermia: a histologic and functional study

The purpose of this study was to evaluate the effect of hyperthermia on the histologic and functional response of the canine kidney, a late-responding normal tissue, to irradiation. Both kidneys were irradiated. Radiation was delivered in single doses of 0, 10, or 15 Gy. Whole-body hyperthermia was used to produce renal kidney temperatures approximating 42.0 degrees C for 60 min. Thirty-six beagles were placed randomly in the following six treatment groups: control, whole-body hyperthermia alone, 10 Gy alone, 10 Gy + whole-body hyperthermia, 15 Gy alone, and 15 Gy + whole-body hyperthermia. Renal histologic and functional changes were assessed at 1 to 9 months after therapy. No changes were seen in glomerular filtration rate or renal tissue volumes in control or hyperthermia alone groups. Renal vascular and glomerular volumes were not affected significantly by any combination of hyperthermia and/or radiation. In all groups receiving radiation, glomerular filtration rate decreased, percentage renal tubular volume decreased, and interstitial volume increased significantly after therapy. The magnitude of these changes in the functional and histologic response of the kidney and the latent period before expression of this damage were dependent on radiation dose. However, hyperthermia did not modify expression of radiation damage in the kidney based on glomerular filtration rate and histologic quantification of renal tissue components.     

Phytochrome regulation of greening in wild type and long-hypocotyl mutants ofArabidopsis thaliana

A brief pulse of red light (R) given to darkgrown seedlings ofArabidopsis thaliana (L.) Heyn. potentiates rapid synthesis of chlorophyll upon transfer to continuous white light. The time course for potentiation of rapid greening shows that a R pulse in the LF (low fluence) range has maximal effect within a few hours, and that there is a small VLF (very low fluence) component as well. Partial reversal of the effect of R by far-red light (FR) indicates that the pulse acts through phytochrome. As it does in the wild-type (WT), a pulse of R accelerates greening of long-hypocotyl (hy) mutants. The extent of induction by the R pulse was about the same in the WT and in allhy mutants studied. Reversibility by FR was greatly decreased in thehy-1 andhy-2 strains. It is possible that these mutants contain a species of phytochrome with defective phototransformation kinetics. If there is such a defective phytochrome species, it nevertheless appears to be active in the potentiation of rapid greening. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Atriopeptin does not augment the transvascular flux of macromolecules in the hamster cheek pouch.

Natriuretic peptides elaborated by atrial myocytes promote marked renal sodium and water excretion as a mechanism for fluid and electrolyte balance. Recent evidence suggests that atriopeptin (ANP) also targets the non-renal vasculature as a site for enhanced fluid exchange. It remains unclear whether ANP alters microvascular integrity to facilitate the efflux of both plasma and proteins across the endothelial barrier, or if fluid exchange is selectively enhanced. This study evaluated the influence of ANP on macromolecular transport through the direct observation of microvessels in the hamster cheek pouch using fluorescent intravital microscopy. Fluorescein isothiocyanate conjugated to either bovine serum albumin or dextran 150,000 Mw was utilized as a permeability probe. Macromolecular efflux was quantified as fluorochrome clearance. The clearance of fluorescein-conjugated bovine serum albumin (57.94 +/- 7.03) or fluorescein-conjugated dextran 150 (4.09 +/- 1.35) remained unaltered by intravascular injection of 1 microgram/kg ANP. Topical application of 40 ng to cheek pouch microvessels produced similar results. All pouches demonstrated positive leakage response to histamine 2.5 x 10(-6) M, increasing fluorochrome clearance approximately 2- to 11-fold. Bolus injection of 1 microgram/kg ANP reduced mean arterial pressure, increased urine flow from 6.63 +/- 2.59 microliters/min to 8.20 +/- 6.13 microliters/min, and elevated sodium excretion from 1.37 +/- 0.49 microEq/min to 2.54 +/- 0.99 microEq/min. These results suggest that ANP fails to significantly alter the integrity of the protein-transporting channels in the microvascular exchange barrier.     

Electrical Energy Changes Conductivity and Determines Optimal Electrotransformation Frequency in Gram-Negative Bacteria

In many bacterial electrotransformation protocols, pulse time is related to the time constant for a capacitor discharging across a sample of fixed resistance. Using an electroporator which controls pulse time independently of the capacitor time constant, we found that the resistance of bacterial suspensions fluctuates widely during capacitor discharge. With three gram-negative species of bacteria, electrotransformation frequency and survival could be more simply related to the electrical energy delivered in each pulse than to component parameters, such as initial field strength, capacitance, and pulse time. In each case, the number of transformants per survivor increased exponentially and leveled off when more than 0.5 to 1.0 J of electrical energy was delivered. An inverse log-linear relationship between survival and energy delivered was also observed for all three species.     

Presence of the bacterial hemoglobin gene improves α-amylase production of a recombinantEscherichia coli strain

A recombinant plasmid (pMK57) was constructed by cloning theBacillus stearothermophilus α-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed intoEscherichia coli strain JM103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced α-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more α-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much α-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.     

Mitochondrial protein import

Most polypeptides of mitochondria are imported from the cytosol. Precursor proteins contain targeting and sorting information, often in the form of amino-terminal presequences. Precursors first bind to receptors in the outer membrane. Two putative import receptors have been identified: a 19-kilodalton protein (MOM19) inNeurospora mitochondria, and a 70-kilodalton protein (MAS70) in yeast. Some precursors integrate directly into the outer membrane, but the majority are translocated through one or both membranes. This process requires an electrochemical potential across the inner membrane. Import appears to occur through a hydrophilic pore, although the inner and outer membranes may contain functionally separate translocation machineries. In yeast, a 42-kilodalton protein (ISP42) probably forms part of the outer membrane channel. After import, precursors interact with chaperonin ATPases in the matrix. Presequences then are removed by the matrix protease. Finally, some proteins are retranslocated across the inner membrane to the intermembrane space.     

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups. Present address: Department of Pathology, Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112 Correspondence to: M.A. Batzer