Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.     

Polyamine Titer in the Embryonic Axis and Cotyledons of Glycine max (L.) during Seed Growth and Maturation

Active polyamine metabolism occurs in Glycine max (L.) seeds during development. Most (≥97%) of putrescine (Put), spermidine (Spd), spermine (Spm), and cadaverine (Cad) are present as free forms in the growing embryo. In the cotyledon or embryonic axis, Put decreases to a nearly undetectable level, while Spd level sharply increases as seed dry weight accumulation progresses. Spm level in the axis also increases along with the Spd level. There is little change in Spm level in the cotyledons. Maturation and dehydration results in a slight reduction of Spd level in the cotyledons. Cad is present in relatively large quantities (5.5-12 micromoles per gram dry weight) in the axes of mature soybean seeds. Only traces of Cad, as expressed on a dry weight basis, are found in the developing or mature cotyledons. The synthesis and accumulation of Cad in the axis begins at the time when the axis or the seed accumulates 30 to 50% of its maximum dry weight. The Cad accumulation (0.8 nanomole per axis per day) proceeds until the later stages of dehydration. When soybean plants are subjected to complete defoliation and shade during the midpoint of seed maturation, Cad accumulation in the axis and seed dry weight accumulation ceased almost immediately. The treatment, however, does not affect the viability of soybean seeds.     

Glyoxylate transamination in intact leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes were supplied with glycolate and one to three of the amino acids serine, glutamate, and alanine, and the amount of the respective α-keto acids formed in glyoxylate transamination was assayed. At 1 millimolar glycolate and 1 millimolar each of the three amino acids in combination, the transamination reaction reached saturation; reduction of either glycolate or amino acid concentration decreased the activity. The relative serine, glutamate, and alanine transamination at equal amino acid concentrations was roughly 40, 30, and 30%, respectively. The three amino acids exhibited mutual inhibition to one another in transamination due to the competition for the supply of glyoxylate. In addition to this competition for glyoxylate, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine. Alteration of the relative concentrations of the three amino acids changed their relative transamination. Similar work was performed with intact oat (Avena sativa L.) leaf peroxisomes. At 1 millimolar of each of the three amino acids in combination, the relative serine, glutamate, and alanine transamination was roughly 60, 23, and 17%, respectively. Similarly, alteration of the relative concentration of the three amino acids changed their relative transamination. The contents of the three amino acids in leaf extracts were analyzed, and the relative contribution of the three amino acids in glycine production in photorespiration was assessed and discussed.     

Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus

Neurons producing growth hormone-releasing factor have been characterized and analyzed by immunohistochemistry in the hypothalami of human fetuses, neonates, infants and adults, using two antibodies against human pancreatic GRF (hpGRF). One of the antibodies recognized both the hpGRF(1-40)OH and hpGRF(1-44)NH2 in the mid portion (between the 28th and 39th amino acid), the other one specifically recognized the C-terminal end of hpGRF(1-44)NH2. These two antibodies stain a single neuronal system with cell bodies mainly located in the infundibular (arcuate) nucleus, and in the ventromedial and lateralis tuber nuclei. These neurons project to the median eminence where they give numerous endings in contact with portal vessels. These neurons are distinct from those containing LH-RH, somatostatin, CRF or pro-opiocortin. In fetuses, neurons immunoreactive with hpGRF antibodies are first detected at the 29th week. They display a neuroblastic aspect which persists after birth. Immunoreactive fibers are detectable in the median eminence after the 31st week. These results demonstrate that the infundibular nucleus plays a major role in control of GH secretion in man and that secretion of GRF appears late during fetal life; this suggests that the first stages of differentiation and development of GH producing cells in the human fetus do not depend on hypothalamic GRF secretion.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Ability of Pollen to Germinate prior to Anthesis and Effect of Desiccation on Germination

The ability of pollen to germinate prior to anthesis was tested using Easter lily (Lilium longiflorum L.) and corn (Zea mays L.). Lily pollen normally dries to a low moisture content between anthesis and pollination while corn does not. The corn pollen germinated well (about 73%) when removed from anthers 1 day before anthesis and placed on culture medium. The lily pollen germinated poorly (0 to 5%) when harvested one to six days before anthesis. However, the lily pollen harvested one or two days before anthesis gave greatly improved germination (about 55%) after it was dried to a low moisture content. The results indicate that an internal control prevents premature germination of lily pollen and that drying is the final stage of pollen maturation. A different sort of regulatory mechanism must operate to prevent premature germination of corn pollen.     

Lysine tRNA and cell division: a G1 cell cycle mutant is temperature sensitive for the modification of tRNA5Lys to tRNA4Lys.

Ts-694 is a temperature sensitive mutant of hamster cells which is blocked in the G1 phase of the cell cycle at the restrictive temperature of 39 degrees. A comparison of the Lys-tRNA isoacceptors by RPC-5 chromatography showed a decrease in tRNA5Lys and an increase in tRNA4Lys at 39 degrees. This was identical to the changes seen in confluent cultures at the permissive temperature of 33 degrees. These Lys-tRNA changes were not seen in ts-694 cells blocked in G1 by isoleucine deficiency, nor in two other G1 ts mutants at the restrictive temperature. Cells trapped in S phase by a thymidine block also contained decreased levels of tRNA4Lys when raised to 39 degrees. Both tRNA4Lys levels and cell division increased when the cells were returned to the permissive temperature. An in vitro assay was established for the modification of tRNA5Lys to tRNA4Lys with tRNA6Lys and tRNA2Lys as intermediates. The first reaction is the synthesis of tRNA6Lys which involves the introduction of a modified uridine at the third position of the anticodon. Extracts of 694 cells grown at 33 degrees were able to modify rat liver [3H] tRNA5Lys to tRNA6Lys and tRNA4Lys in vitro when assayed at 25 degrees but not at 39 degrees. Extracts of Balb/c 3T3 cells, however, were more active at 39 degrees than at 25 degrees showing that the normal enzyme is not temperature sensitive. Ts-694 cell tRNA, isolated from cells grown at 33 degrees was aminoacylated at both 25 degrees and 39 degrees with rat liver synthetases. tRNA4Lys was present at both temperatures indicating that ts-694 cells do not contain a temperature sensitive tRNA4Lys.     

Circular-dichroic studies on the conformational behaviour of troponin and tropomyosin from bovine cardiac muscle.

Bovine cardiac troponin is similar to rabbit skeletal troponin with respect to secondary structure, amino acid composition and molecular weight of the subunits, but differs slightly with respect to biological activity and surface charges of the subunits. Previous circular-dichroic studies of the subunits and recombination of subunits have indicated significant Ca2+-induced delocalized conformational changes. Present studies of the native troponin complex are not in accord with such changes. Furthermore the formation of the troponin-tropomyosin complex in vitro results in no delocalized conformational changes, nor does it sensitize troponin to Ca2+-induced changes. It is suggested that the troponin complex cannot be dissociated into subunits without significant and irreversible conformational perturbation.