Stimulation of coho salmon growth by insulin-like growth factor I.

The effect of insulin-like growth factor I on growth rate of coho salmon (Oncorhynchus kisutch) was examined. Juvenile coho salmon received implants of osmotic minipumps containing recombinant bovine insulin-like growth factor I (rbIGF-I) or saline for a period of 3 to 4 weeks. High doses of rbIGF-I (greater than 0.13 microgram.g-1.d-1) resulted in hypoglycemia and death. In 2-year-old coho salmon, 0.09 microgram.g-1.d-1 rbIGF-I administered for 25 days doubled linear growth rate and increased growth rate in weight by 40%. In rapidly growing, 1-year-old coho salmon, growth rate was not altered by rbIGF-I at 0.01 or 0.05 micrograms.g-1.d-1 for 31 days. In ration-limited fish exhibiting slow growth in the control group, rbIGF-I (0.02 microgram.g-1.d-1) increased linear growth rate by up to threefold and growth rate in weight by up to fourfold. The results indicate that exogenous treatment with mammalian IGF-I can stimulate coho salmon growth under some conditions, and that endogenous IGF-I may be an important factor in regulating growth of teleosts.


Neuropeptide Y-like immunoreactivity in the dogfish gastroenteropancreatic tract: light and electron microscopical study.

The 36 amino acid neuropeptide Y (NPY) has been examined in mammals and is mainly located in the nerves. Its distribution in nonmammalian vertebrate and in some invertebrate nervous systems has been confirmed. Using antisera raised to porcine NPY, NPY immunoreactivity has been localized in endocrine cells of the pancreas and gastrointestinal tract of two dogfish, Scyliorhinus stellaris and Scyliorhinus canicula. Immunostained serial sections and cross-absorption experiments with related peptides, including avian and bovine pancreatic polypeptide and peptide tyrosine tyrosine, excluded any cross-reactivity. The fine structure of the cells containing NPY-like substance is described.


Regulation of calbindin D 28K and its mRNA in the intestine of the domestic hen.

Intestinal calbindin synthesis in laying hens was analyzed to assess controlling factors operating during egg formation. In the absence of vitamin D, calbindin was not induced by estrogen and testosterone. In immature vitamin D-replete pullet, blood levels of 1,25(OH)2D3 increased in response to estrogen but the duodenal concentration of calbindin and its mRNA were increased only when testosterone was given together with estrogen. The plasma concentration of 1,25(OH)2D3 and the duodenal levels of calbindin and its mRNA were substantially higher in laying hens than in immature pullets. No differences in these parameters were observed between the stages of the ovulatory cycle. Suppression of shell formation for a week decreased the concentration of 1,25(OH)2D3 and of duodenal calbindin but did not affect the level of its mRNA. When egg shell formation resumed in hens previously laying shell-less eggs, the concentrations of 1,25(OH)2D3 and of calbindin and its mRNA increased toward the end of shell formation. A most important factor regulating intestinal calbindin synthesis in laying hens turned out to be 1,25(OH)2D3. Intestinal calbindin mRNA is more stable in laying hens than in young birds as its concentration declines more slowly when the stimulation provided by 1,25(OH)2D3 is withdrawn, as occurs following suppression of shell formation and after parathyroidectomy in laying birds. Intestinal calbindin mRNA is therefore increased by a process other than increasing 1,25(OH)2D3 formation. The factor influencing the stability of this mRNA in laying hens could be calcium. It is concluded that in hens the increased duodenal calbindin synthesis elicited by plasma 1,25(OH)2D3 at sexual maturity primarily involves a transcriptional process and the stabilization of the mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of a sustained-release estrogen implant on oviduct development and plasma Ca concentrations in broiler breeder chicks: modulation by feed restriction and thyroid state.

The effect of estradiol-17 beta (E2) given as a sustained-release implant (Compudose 200) on concentrations of plasma calcium (Ca) and the development of the chick shell gland has been investigated in food-restricted and thyroid hormone-treated 6- to 8-week-old broiler breeder pullets. Chicks implanted with 0, 0.5, 1.0, 1.5, 2.0, and 3.0 Compudose pellets for 24 days (n = 6/group) revealed a dose-response relationship between plasma E2 and Ca and on oviduct growth. Plasma E2 concentrations were characterized by an initial burst phase for approximately 17 days, followed by a constant release phase. Histologic examination of shell gland tissue confirmed the dose related E2-induced development of microvilliated epithelium and tubular glands over time. Feed restriction initiated at 2 weeks of age markedly increased the response to the E2 implants. Birds (n = 8/group) implanted with 2 pellets and feed restricted had increased plasma concentrations of E2 and Ca, and increased growth of the oviduct (P less than 0.01) as compared to ad libitum implanted birds. In a separate study birds (n = 6/group) had restricted access to feed from 8 weeks of age and were implanted with 0, 2, 4, or 8 pellets. At intervals from 9 to 45 days after implantation one bird from each group was killed. Although concentrations of plasma Ca were significantly greater in feed-restricted birds (P less than 0.01), oviduct growth was only marginally increased by the food restriction program. Plasma Ca concentrations in broiler breeder pullets (n = 8/group) implanted with 1 or 3 pellets and injected with T3/T4 (100 micrograms/day) were significantly decreased (P less than 0.05). Injection of thyroid hormone also marginally decreased shell gland epithelial cell height (P less than 0.05) and development of microvilli (P less than 0.05). There was no effect of the administration of the goitrogen, propylthiouracil (10 micrograms/day im), on the E2 induced development of the shell gland.


A reduction in pituitary dopamine turnover is associated with sex pheromone-induced gonadotropin secretion in male goldfish.

In goldfish, the gonadal steroid, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), functions as a potent preovulatory female sex pheromone which stimulates rapid elevations in serum gonadotropin (GtH) levels and subsequent increases in milt production in males. GtH secretion in goldfish is known to be regulated by the stimulatory actions of gonadotropin-releasing hormone (GnRH) and the inhibitory actions of dopamine (DA). This study specifically examined whether the 17,20 beta-P-induced elevation in male GtH is caused by pheromone-mediated changes in DA inhibition at the level of the pituitary. First, we have demonstrated that dihydroxyphenylacetic acid (DOPAC) is the primary metabolite of DA catabolism in the brain and pituitary gland of goldfish. Second, we measured changes in circulating levels of GtH and changes in pituitary content of DA and its metabolite, DOPAC, as well as possible alterations in DA turnover rate (DOPAC/DA ratio) following short-term exposure of male goldfish to water-borne 17,20 beta-P. Water-borne 17,20 beta-P consistently increased serum GtH levels in males within 20 min of exposure and maintained elevated levels for up to 120 min. Although changes in pituitary DA content were not observed during periods of high GtH release, coincident reductions in pituitary levels of DOPAC were measured within 45 min of exposure to the pheromone. More importantly, there was a significant decrease in the rate of DA turnover in the pituitary, as assessed by comparing the ratio of DOPAC to DA present, at 20, 45, and 120 min of exposure. Since the reduction of DA turnover in the pituitary is inversely correlated with periods of increased GtH release, the present results suggest that water-borne 17,20 beta-P causes an abatement of DA release to the pituitary. Based on the latency of the GtH response to water-borne 17,20 beta-P, a rapid reduction of DA turnover in the pituitary appears to be at least part of the neuroendocrine trigger for 17,20 beta-P-induced GtH release in male goldfish.


Structure-cardiovascular activity relationships of parathyroid hormone.

The effects of parathyroid hormone (PTH) analogues on isolated rat tail arteries and frog arteries and heart were examined. Synthetic bovine PTH fragment 1 to 34 [bPTH-(1-34)], porcine PTH-(1-34), [Ala1] pPTH-(1-34), and [Nle8] pPTH-(1-34) were tested for vasorelaxant activity on helical strips of the rat tail artery and the frog iliac and femoral artery, preconstricted with arginine vasopressin or arginine vasotocin in the rat and the frog, respectively. For cardiac activity, PTH analogues were administered to isolated frog atria, and atrial rate and atrial tension (AT) were measured. The N-terminal amino acid alanine appears to be pivotal for vasorelaxant activity of PTH molecules on the rat tail artery strips. Data from the frog preparations supported the finding in the rat, although the iliac and femoral arterial preparations differed in their responses. The N-terminal serine in pPTH-(1-34), compared with the other PTH analogues, appeared to be responsible for different cardiac responses, being negatively chronotropic and without effect on AT.


A possible involvement of prostaglandin F2 alpha (PGF2 alpha) in Rana esculenta ovulation: effects of mammalian gonadotropin-releasing hormone on in vitro PGF2 alpha and 17 beta-estradiol production from ovary and oviduct.

The present work studied the PGF2 alpha and 17 beta-estradiol plasma levels during ovulation, and the in vitro effects of mammalian gonadotropin-releasing hormone (mGnRH) on ovarian and oviductal production of prostaglandin F2 alpha (PGF2 alpha) and 17 beta-estradiol during four different stages of the annual sexual cycle in water frog, Rana esculenta. Plasma levels of PGF2 alpha increased in ovulating frogs, with respect to preovulatory and postovulatory levels, while estradiol did not change. In addition, mGnRH increased PGF2 alpha and 17 beta-estradiol in the incubation media of ovaries taken during the recovery stage. Moreover, mGnRH increased PGF2 alpha in incubation media of oviducts collected during the reproductive stage. These findings suggest that PGF2 alpha could be involved in the control of egg deposition in the female R.


Plasma progesterone levels in the pregnant female rat-kangaroo (Bettongia gaimardi).

Plasma progesterone levels were measured in female bettongs (small macropodid marsupials) under two natural regimes: (1) during "delayed" gestation (initiated by removal of pouch young, RPY) and (2) during the pregnancy prior to pouch vacation when a young still occupies the pouch (i.e., during lactation). Basal levels of progesterone were 0.15-0.5 ng/ml. There was a transient peak of progesterone (0.7 ng/ml) early in gestation at Day 4 RPY. After Day 6 RPY, progesterone levels remained elevated (1.2-1.5 ng/ml) until they dropped sharply to basal levels on the day of birth. This pattern of progesterone secretion during delayed gestation is similar to that seen in other marsupials, such as the tammar wallaby. There was no significant difference between the progesterone profiles of the two experimental groups. We deduce, then, that lactation had no effect on corpus luteum function (as assessed by plasma progesterone levels) in the pregnant bettong.


Lack of effect of TRH on alpha-MSH release from the neurointermediate lobe of the lizard Lacerta vivipara.

Thyrotropin-releasing hormone (TRH) is a potent stimulator of melanotropin (alpha-MSH) release from pituitary melanotrophs in pig, frog, and fish. Concurrently, it has recently been shown that injection of TRH induces skin darkening in the lizard Anolis carolinensis (Licht and Denver, 1988). In the present study, we have thus investigated in vitro the possible effect of TRH on alpha-MSH release from the lizard (Lacerta vivipara) neurointermediate lobe, by means of the perifusion technique. Using our radioimmunoassay procedure, we found that serial dilutions of L. vivipara NIL extracts and synthetic alpha-MSH gave parallel binding curves. Administration of graded doses of TRH (10(-8)-10(-6) M) did not cause any modification of alpha-MSH release. In contrast, infusion of a depolarizing concentration of K+ induced a robust stimulation of alpha-MSH secretion. These results indicate that, in the lizard L. vivipara, the neuropeptide TRH does not stimulate pituitary melanotrophs.


In vitro effects of homologous prolactins on testosterone production by testes of tilapia (Oreochromis mossambicus).

The tilapia, Oreochromis mossambicus, produces two prolactins designated tPRL177 and tPRL188 to indicate the number of amino acid residues in each. The direct in vitro actions of tPRL177 and tPRL188 on basal and ovine luteinizing hormone (LH)-induced testosterone production in minced testes of courting and noncourting (bachelor) tilapia were examined. Courting males were housed with females and were used as soon as they built a spawning pit; noncourting males were housed with other males and were used when they appeared female-like in coloration. The in vitro culture system consisted of two phases. In phase 1 (0-10 hr), testicular tissue was incubated without (control) or with the tPRLs--both alone and together and in doses ranging from 0.5 to 32 microM. In phase 2 (10-16 hr), the culture medium was replaced with medium containing 6.25 microM (0.25 micrograms/ml) ovine LH. Testosterone production during both phases was determined by radioimmunoassay of the medium. The tPRLs stimulated testosterone production during phase 1 in courting males (by 25%) but not in noncourting males. Exposure to either or both tPRLs in phase 1 enhanced the response of testicular tissue from courting males to ovine LH (by 10%) and inhibited the response of minced testes taken from noncourting males (by 12%). This is the first evidence to demonstrate direct gonadal activity of homologous PRL in a teleost.


Inhibition of protein kinase C antagonizes in vitro tadpole tail fin regression induced by thyroxine.

Tail fin regression can be induced in anuran amphibians with L-thyroxine (T4). This regression can be antagonized with prolactin (PRL). Previous work had suggested that protein kinase C (PKC) was involved in PRL action. To address this issue further, the effect of a potent and selective inhibitor of protein kinase C on in vitro tail fin regression was investigated. T4-induced regression of tail fin pieces from Rana pipiens tadpoles could be antagonized by adding PRL or the PKC inhibitor H-7 to the medium. H-7 inhibited fin regression in a dose-dependent manner, with a half-maximal effective concentration of about 10(-5) M. The H-7 analogue, HA-1004 (which is not a selective inhibitor of PKC), was without effect. These results suggest a possible role for PKC in tail fin regression and may be useful in elucidating the antimetamorphic action of PRL.


Effects of nutritional state, insulin, and glucagon on lipid mobilization in rainbow trout, Oncorhynchus mykiss.

The effects of nutritional state, insulin, and glucagon on lipid mobilization were determined in rainbow trout, Oncorhynchus mykiss. In nutritional state experiments, fish were either fed continuously (except 24 to 36 hr prior to experimentation) with commercial trout chow or fasted for 4 weeks. Lipase activity in liver tissue isolated from fasted fish and cultured for 5 hr was greater than that in tissue isolated from fed fish and cultured. The presence of glucose (5.55 mM) in the incubation medium accentuates lipolytic activity in both liver and adipose tissue. Hormone response was assessed both in vivo and in vitro. Salmon insulin was injected into anesthetized fish (fed continuously except 24 hr prior to injections) in 10 microliters of saline/g body weight; final hormone dose was 100 ng/g body weight. Tissue and plasma were sampled 1 and 3 hr after injection. Insulin resulted in depressed plasma FA concentration and reduced hepatic triacylglycerol lipase activity. In vitro effects of hormones were evaluated by incubating liver and adipose tissue pieces in Hanks-MEM. Glucagon (bovine/porcine) directly stimulated lipid breakdown in both liver and adipose tissue. These actions were manifested by enhanced FA and glycerol released into the culture medium and by elevated triacylglycerol lipase activity. Insulin (bovine) generally appeared antilipolytic as this agent inhibited glucagon-stimulated lipase activity and glucagon-stimulated FA release. Furthermore, insulin (in the presence of glucose) reduced net lipolysis, as indicated by glycerol release, compared to control cultures. These results indicate that nutritional state and glucose are important modulators of lipid mobilization and that glucagon and insulin act directly on lipid storage sites to coordinate lipolysis in rainbow trout.


Liver and brain glucocorticoid receptor in rainbow trout, Oncorhynchus mykiss: down-regulation by dexamethasone.

Total glucocorticoid binding sites were identified and quantitated in liver and brain of rainbow trout using an exchange method and [3H]dexamethasone as the ligand. Both tissues contained a predominantly cytosolic moiety that bound dexamethasone with high specificity. Binding was saturable, time dependent, and completely reversible. Scatchard analysis showed a linear relationship suggesting that receptors belong to a single class. Dexamethasone down-regulated both liver and brain receptors. Down-regulation was rapid (within hours) and dose dependent (ED50 = 1.5 mg/kg body weight). Dexamethasone-induced down-regulation was not a result of cytoplasm to nuclei translocation or due to increases in tissue concentrations of steroid. Dexamethasone administration resulted in a lowering of Bmax (82.3 +/- 2.5 to 20.6 +/- 10.5 fmol/mg protein) and an increase in Kd (15.6 +/- 0.2 to 44.3 +/- 5.0 nM) suggesting a conformational change in the receptor molecule as part of the mechanism. The brain and liver of the rainbow trout thus have glucocorticoid receptors similar to those described in the mammalian system. Further, these receptors are subjected to autologous regulation similar to their counterparts in other systems.


Plasma sex steroid binding proteins (SSBP) in the male lizard, Podarcis s. sicula, during the reproductive cycle.

In male Podarcis s. sicula plasma, a sex steroid binding protein [SSBP(s)] binds testosterone (T) and estradiol-17 beta (E2) with moderate affinity (Kd = 0.23 +/- 0.08 x 10(-8) for 3H-E2, and 0.24 +/- 0.07 x 10(-8) for 3H-T) and high capacity. The SSBP binding affinity is unchanged throughout the sexual cycle, although its capacity is higher in nonreproductive males (winter and postreproductive period). This change may be related to changes in plasma T and E2 levels, and is likely to be involved in mechanisms whereby free steroid is delivered to target organs. SSBP, under isoelectrofocusing, is distributed between pH 5.5-6.5 and pH 7.1-7.5. The concentration of these two forms varies during the annual cycle.


Mammalian GnRH involvement in prostaglandin F2 alpha and sex steroid hormones testicular release in two amphibian species: the anuran water frog, Rana esculenta, and the urodele crested newt, Triturus carnifex.

The present work was carried out to study the in vitro effects of mammalian gonadotropin-releasing hormone (mGnRH) on Rana esculenta and Triturus carnifex testis production of prostaglandin F2 alpha (PGF2 alpha) and sex steroid hormones during the prereproduction, reproduction, and postreproduction periods. In R. esculenta, testicular PGF2 alpha release was lower during postreproduction, and mGnRH increased PGF2 alpha in prereproduction and reproduction. Androgens were higher during prereproduction, and mGnRH induced an androgens increase in prereproduction and reproduction. In T. carnifex testicular PGF2 alpha release was lower during reproduction, and mGnRH increased PGF2 alpha in prereproduction and reproduction. Androgens were higher in reproduction and lower in postreproduction, and mGnRH induced an androgens increase in reproduction. Estradiol-17 beta release was higher in postreproduction, and mGnRH induced an estradiol decrease in reproduction and an increase in postreproduction. These results seem to indicate the involvement of PGF2 alpha in the testicular reproductive activity, and a similar mGnRH mechanism of action, both in R. esculenta and in T. carnifex. In addition, taken together with previous studies, they seem to suggest that the relationship found between mGnRH and PGF2 alpha or sex steroids could be widespread in amphibians.


Molecular cloning of silver carp and bighead carp prolactin.

The cDNAs encoding the prolactin of silver carp (scPRL) and bighead carp (bcPRL) have been cloned. Deduced from the nucleotide sequences, both scPRL and bcPRL are composed of 187 amino acid residues. Only one residue is different between scPRL and bcPRL. Homology analysis indicates that scPRL and bcPRL are highly homologous to carp PRL (97%), relatively conserved in relation to PRLs of salmon, trout, and tilapia (64-69%), and diversified from avian and mammalian PRL (30-35%). Similar to PRLs of other species of fish, scPRL and bcPRL lack the first 12 N-terminal residues of avian and mammalian PRLs.


Gonadotropin-releasing hormone from thai catfish: chromatographic and physiological studies.

Two forms of immunoreactive gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of Thai catfish, Clarias macrocephalus and C. batrachus. The peptides were detected using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). In both the HPLC systems, catfish GnRH-I eluted earlier than catfish GnRH-II and also eluted before the synthetic standards of mammalian, lamprey, chicken I, chicken II, and salmon GnRH. Hence, catfish GnRH-I appears to be the most hydrophilic GnRH family member because of this early elution from the HPLC. Catfish GnRH-II eluted in a position similar to that of chicken GnRH-II. This study suggests that catfish GnRH-I is a novel form of GnRH, whereas catfish GnRH-II is the same as chicken GnRH-II. Indirect evidence suggests that the catfish molecule is 10 amino acids in length and has an amide at the C-terminus. Moreover, the novel catfish GnRH appears to be different within the domain of amino acids 5 to 10 compared with mammalian GnRH because it is not recognized by antiserum B-6. An injection of native chicken GnRH-II was more effective than salmon or mammalian GnRH for induced ovulation in C. macrocephalus.


Isolation and amino acid sequence of urotensin II from the sturgeon Acipenser ruthenus.

Urotensin II (UII) peptides have previously been isolated from the urophysis (the neurohemal organ of the caudal neurosecretory system) of several teleost fish, and the UII amino acid sequences have been determined. Chondrostean fish, such as the Acipenseridae (sturgeon), though without a distinct urophysis, also have a caudal neurosecretory system, which has been indicated by bioassay and immunological evidence to contain UII-like peptides. In the present studies, we investigated by UII radioimmunoassay the UII-like peptides in the spinal cord of three Acipenser species, and isolated and sequenced UII from one of them. As expected, much more UII immunoreactivity (UII-IR) was found in caudal than in anterior spinal cord extracts. In addition, caudal extracts from A. ruthenus were found to contain much more UII-IR (whether determined on a UII-IR/weight or UII-IR/fish basis) than those from the larger A. stellatus and A. guldenstadti. UII was therefore isolated from A. ruthenus and its amino acid sequence was shown to be H-Gly-Ser-Thr-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. This sequence is identical at positions 6-11 (the disulfide ring) with the known teleost UII peptides, and has acidic and hydrophobic amino acids at positions 5 and 12, respectively, as do the teleost UII peptides. Overall sequence identity with the various forms of teleost UII was 58-83%.


Changes of LH level in the pituitary gland and plasma in hibernating frogs, Rana temporaria.

Pituitary and plasma luteinizing hormone (LH) levels were measured in female frogs, Rana temporaria, during and immediately after hibernation (0-4 degrees in darkness; 22-25 weeks) to study regulation of gonadotropin leading to posthibernation ovulation. Pituitary LH content began to rise progressively during the last third of hibernation (e.g., from starting levels of 5.3 +/- 0.09 micrograms/gland, 1.5 micrograms/mg between 8 and 11 weeks, to 18.6 +/- 9.7 micrograms/gland, 5.0 +/- 2.7 micrograms/mg at 19-22 weeks). Plasma LH increased in parallel, but with some delay (from 10 +/- 8 to 25 +/- 24 ng/ml). Frogs kept in light at low temperatures showed similar responses. Release of LH (rise in plasma levels with a drop in pituitary content) was observed during 32 hr immediately after termination of hibernation in association with the onset of ovulation. These data indicate the existence of regulatory mechanisms operating during hibernation under conditions of constant cold. Altogether, these and our previous results obtained after surgical deafferentiation of median eminence support the hypothesis of a progressive reduction of a CNS inhibition that results in the release of GnRH during hibernation in this frog.


Progesterone metabolism in vitro in the decapod crustacean, Penaeus monodon.

Thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) with on-line detection of radioactive steroids were applied to identify metabolites of [4-14C]progesterone incubated in vitro with prawn ovary. There was extensive metabolism of progesterone by stage II (vitellogenic) ovary of Penaeus monodon. The most abundant metabolites were 5 alpha-pregnane derivatives together with two minor metabolites, 20 alpha-hydroxypregn-4-en-3-one and 1,4-pregnadiene-3,20-dione. In contrast, a much lower level of progesterone metabolism was observed in stage 0 (immature) ovary of this species. The hepatopancreas, gill, and abdominal muscle of P. monodon all metabolised [4-14C]progesterone to varying degrees, generating materials similar to those produced by the ovary. A comparative study of progesterone metabolism in stage II ovary of Nephrops norvegicus indicated that one metabolite, 20 alpha-hydroxypregn-4-en-3-one, was produced.


Growth and behavior of thyroid-deficient lizards (Sceloporus undulatus).

This study investigates thyroid control of growth and energy metabolism plus growth-dependent and growth-independent behavioral effects of thyroid manipulation in lizards. Experiments were done on surgically thyroidectomized (Tx) and sham-operated (Sh) yearling Sceloporus undulatus enclosed in their natural habitat. Lizards were placed in an outdoor enclosure in early August. Growth rate was measured and behavior was observed until mid-October. Subsequently, lizards were returned to the lab for measurements of plasma thyroxine (T4), standard metabolic rate (SMR), and running endurance. Thyroidectomy reduced plasma T4 from 4.3 +/- 0.56 ng/ml to undetectable levels (P = 0.006) and SMR by 44% (P less than 0.0001). Thyroid deficiency produced a threefold reduction in growth rate (Tx: 0.04 +/- 0.010 mm/day, n = 12 vs Sh: 0.11 +/- 0.006 mm/day, n = 11, P less than 0.0001). Growth rate was correlated with SMR among individuals (length-specific: r = 0.55, P = 0.027, n = 16), even after statistical removal of mass and treatment effects. The total volume of oxygen consumed for standard metabolism during the growth period, as estimated from allometric equations, was correlated with cumulative growth (R2 = 0.94, P = 0.013) and was significantly lower for thyroid-deficient lizards than for controls (P less than 0.0001). Distance moved in the field and running endurance on a treadmill both scaled on body mass (M0.98 +/- 0.418, P = 0.030 and M1.72 +/- 0.763, P = 0.040, respectively), indicating that larger lizards moved farther and had greater stamina than their smaller counterparts. Neither of these behavioral factors was affected directly by thyroid status. Thyroid-deficient lizards were active for a smaller percentage of the day than controls (Tx: 42.6 +/- 5.7%, n = 11 vs Sh: 57.4 +/- 3.2%, n = 12, P = 0.040), independent of differences in body size. On an interindividual basis, the frequency of activity was significantly correlated with SMR (r = 0.57, P = 0.020, n = 16) and growth rate (mass-specific: r = 0.47, P = 0.025, n = 23).


Stably inherited killer activity in industrial yeast strains obtained by electrotransformation.

Killer-sensitive strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis were transformed by electroinjection using double-stranded RNA isolated from a superkiller strain. Various recipient strains were used: both thermo-resistant and thermo-sensitive as well as mutants of industrial strains. Conversion of respiratory competent (rho+) into respiratory deficient (rho-) strains (mutants) resulted in a significant increase of the yield of electrotransformants and/or of longterm killer stability. Electrotransformation of rho- mutants of distillery and brewery strains resulted in more than 100 clones, which exhibited weak or strong killer activity over some or all of the experimental period of 10 months.


The serotyping of Azospirillum spp. by cell-gold immunoblotting.

Ten Azospirillum strains were serotyped using the method of cell-gold immunoblotting (dot-blot immune-overlay assay). Colloidal gold-protein A conjugate was used. Antibodies raised against the whole cells showed strain specificity and interacted mainly with carbohydrate antigens on the cell surface. Immunological identity for A. brasilense Sp 245 and Sp 107 strains was found. Cell-gold immunoblotting can be recommended for serotyping of a wide variety of bacterial strains.


Esterase electrophoresis compared with biotyping for epidemiological typing of Acinetobacter baumannii strains.

Forty-nine Acinetobacter baumannii strains belonging to three biotypes and isolated from four hospitals were differentiated by electrophoretic typing of their esterases. Six main kinds of esterases were distinguished by their spectra of hydrolytic activity toward seven synthetic substrates. The electrophoretic variations of these enzymes were used to define ten zymotypes among the three biotypes. Esterase electrophoresis appeared to be more sensitive than biotyping, and could represent an additional marker for epidemiological analysis.


Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis.

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.


Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis.

Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B. thuringiensis. In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups. Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes. In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation. These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.


Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli.

We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating cells of Escherichia coli. Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to betaine uptake. Our data suggest that E. coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites. The ProU system of E. coli, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake.


Serogroups of Escherichia coli strains producing cytotoxic necrotizing factors CNF1 and CNF2.

The serogroups of 396 necrotizing Escherichia coli of human and bovine origin isolated in Spain between 1979 and 1991 have been determined. The 270 cytotoxic necrotizing factor strains belonged to 22 different O serogroups; however, 84% (226 of 270) were of one of seven serogroups (O2, O4, O6, O14, O22, O75 and O83). Although necrotizing E. coli producing cytotoxic necrotizing factor 2 belonged to 28 different serogroups, only six of them (O1, O3, O15, O55, O88 and O123) accounted for 60% (76 of 126) of cytotoxic necrotizing factor 2 strains. Furthermore, only 3% (4 of 126) of cytotoxic necrotizing factor 2 strains belonged to serogroups most common among strains producing cytotoxic necrotizing factor 1. The majority of necrotizing E. coli producing cytotoxic necrotizing factor 1 were obtained from human extraintestinal infections, whereas cytotoxic necrotizing factor 2 strains were isolated from stools of healthy and diarrhoeic calves.


Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase.

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.


Cloning and characterization of an amplified DNA sequence in chromosomal DNA of Streptomyces aureofaciens 2201.

In strain 2201 of Streptomyces aureofaciens, a high copy number amplified DNA sequence (ADS-Sa2201) was found and characterized. The amplified sequence in the chromosomal DNA of this strain forms a stretch of about 10 kb tandemly repeated 100-500 times. In this strain also, extrachromosomal copies of the repeated unit of the ADS-Sa2201 were found. In cloning experiments any autonomous replicon was found on ADS-Sa2201 and it thus can be presumed that the presence of the extrachromosomal copies of the repeated unit of ADS-Sa2201 is only a result of its excision from the chromosome. A part of the repeated unit of ADS-Sa2201 was also found in chromosomal DNA of strain 13 of S. aureofaciens. In this strain no part of ADS-Sa2201 is present extrachromosomally.


Hydrogenase mutants of Alcaligenes eutrophus H16 show alterations in the electron transport system.

Mutations in the genes coding for the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus strain H16 significantly affected the expression of respiratory chain components. In lithoautotrophically grown wild type cells electron flow mainly proceeded via the cytochrome c oxidases. Mutants defective in the membrane-bound hydrogenase contained a 2- to 3-fold higher cytochrome a content than the wild type and cytochrome c oxidase of the aa3-type was preferentially used by these cells for substrate oxidation. Mutants impaired in the soluble hydrogenase revealed slow growth on hydrogen, presumably due to inefficient reverse electron flow mechanisms which provide the cells with NADH for autotrophic CO2-fixation. In this class of mutants the two quinol oxidases of the o- and d-type in addition to the co-type oxidase were the predominant electron-transport branches.


Characterisation of a novel repetitive DNA sequence from Mycobacterium bovis.

We report characterisation of three copies of a novel repeat sequence isolated from a Mycobacterium bovis genomic library. The repeat occurs within open reading frames, potentially encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-X-Gly. The tandem array is present up to five times in M. bovis and it is proposed that they may occur in a family of genes expressing functionally related proteins. We postulate that these proteins may play a role in binding of M. bovis to host cell receptors.


Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species.

A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.


Cosmid pJAR4, a novel Streptomyces-Escherichia coli shuttle vector for the cloning of Streptomyces operons.

A novel shuttle cosmid vector (pJAR4), based on pK505, was constructed for the cloning of Streptomyces DNA. It is a low-copy-number vector which determines hygromycin B-resistance as a selective marker and was used to clone the puromycin biosynthesis pathway from Streptomyces alboniger. Cosmids pJAR4 and pKC505 (which determines apramycin-resistance) stably co-transform both Streptomyces lividans and Streptomyces griseofuscus.


Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media.

Anodic stripping voltammetry of bacterial growth medium containing copper(II) and ampicillin shows that Cu(II) is complexed by the antibiotic and that this complex decomposes to give Cu(II) complexes with ligands derived from ampicillin. At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition. The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II); the unexpected growth of the ampicillin-sensitive host E. coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin. The instability of ampicillin (and other beta-lactam antibiotics) to metal ion-catalysed hydrolysis means that great care must be taken to ensure that such reactions do not occur in growth media. Furthermore, it is clear that double selection for resistance to ampicillin and metals such as Cu(II), Zn(II), Co(II) and Cd(II) is impossible.


Heterotrophic sulfur reduction by Thermotoga sp. strain FjSS3.B1.

Thermotoga sp. strain FjSS3.B1 was able to reduce sulfur to sulfide when grown on a mineral medium with glucose as the sole carbon and energy source. There was no increase in specific growth yield coupled to sulfur reduction, but the specific growth rate, final growth yield, and tolerance of H2 were all increased in the presence of sulfur. At dissolved H2 concentrations, of 550 to 600 mumol/l (at 77 degrees C) growth was not possible unless sulfur was added. Glucose was fermented via the Embden-Meyerhof-Parnas pathway to lactate, acetate, H2 and CO2 (and other unidentified minor products). The thermodynamic problems associated with the relatively high redox potential electrons from the 1,3-bisphosphoglycerate/glyceraldehyde 3-phosphate couple (E'0 = -350 mV) are overcome by reducing sulfur to sulfide (E'0 = -270 mV) rather than the energetically unfavourable production of H2 (E'0 = -414 mV). Under high hydrogen partial pressures there was increased production of lactate as an alternative electron sink. The results indicate that sulfur reduction operates primarily as an electron sink rather than as a detoxification reaction or energy-generating mechanism.


Sequence of the gene encoding type F neurotoxin of Clostridium botulinum.

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.


Tris-dependent site-specific cleavage of Streptomyces lividans DNA.

During conventional gel electrophoresis, Streptomyces lividans DNA undergoes site-specific double-strand cleavage at the positions of closely opposed unstable modifications introduced into the DNA in vivo. We investigated this electrophoretic instability and demonstrated that it was dependent on Tris. Tris buffer was activated at the anode to generate a nucleolytic species; prior to activation, Tris was not able to cleave the DNA. The nucleolytic species was shown to react with thiourea, which could thus protect the DNA from strand cleavage. Non-degradative electrophoresis of the DNA could also be achieved in an alternative buffer such as Hepes.


Metabolism of 3-methyldiphenyl ether by Sphingomonas sp. SS31.

The bacterium Sphingomonas sp. SS31, which was obtained from the diphenyl ether-degrading strain Sphingomonas sp. SS3 by an adaptation process, utilized 3-methyldiphenyl ether for growth in addition to diphenyl ether. The initial enzymatic attack onto this compound proceeded by a regioselective, but non-specific dioxygenation at the carbon carrying the ether bridge and the adjacent carbon of the unsubstituted as well as the methyl-substituted aromatic nucleus. Upon spontaneous decomposition, the resulting unstable hemiacetal structure yielded 3-methylphenol and catechol, or phenol, 3-methylcatechol, and 4-methylcatechol, respectively. Phenol and 3-methylphenol were oxidized to the corresponding catechols which, after subsequent ortho-cleavage, were channeled into the oxoadipate pathway.


Pedigree analysis of blood pressure in subjects from rural Greece and relatives who migrated to Melbourne, Australia.

Diastolic blood pressure readings taken in 1983-1984 on 1,474 Greek individuals (628 living on the island of Levkada, 846 relatives having migrated to Melbourne, Australia) from 204 two generational pedigrees were analysed. Blood pressure was regressed as a quadratic in age by sex and migrant status, and on temperature. Variance increased with age and was greater in migrant males. The covariance between relatives in different countries was significant. Variation was modeled by a multivariate normal model for pedigree analysis in terms of genetic effects, a common environment effect, and effects particular to an individual. The genetic component was 25.9 mm Hg2, independent of sex and migrant status. Importantly, the common environment component was not significant. The third component was greatest in migrant males. Spouse correlation was -0.09 (SE = 0.03). Exclusion of 86 individuals who reported currently receiving medication for elevated blood pressure stabilised the variance and decreased the genetic component. The data suggest that familial aggregation of diastolic blood pressure is due to genetic factors which produce the same variation in males and females, living on Levkada or in Melbourne. Nongenetic factors explain the greater variation in blood pressure of migrant males living in Melbourne.


Estimation of the frequency of isoform-genotype discrepancies at the apolipoprotein E locus in heterozygotes for the isoforms.

Estimates of the impact of apolipoprotein E (apo E) alleles coding for the three common isoforms on plasma lipid levels assume genetic homogeneity among the genotype classes. To test this assumption, we have determined the apo E genotype at the two common polymorphic sites (amino acids 112 and 158) by DNA amplification and hybridisation with allele-specific oligoprobes, in 195 unrelated Caucasian participants of the Rochester Family Heart Study previously classified as heterozygotes by isoelectric focusing (IEF). Fourteen discordant samples were initially detected. Repeat typing of these samples by both methods resolved nine discrepancies and analysis of additional blood samples from the remaining five individuals eliminated a further four discrepancies. The only truly discordant allele was found in a female subject who had an E3 isoform with the common E2 (Cys112, Cys158) genotype. Transmission of this allele from the mother was demonstrated. From these results, we estimate the frequency of discrepancies between isoforms and common genotypes to be 0.25% in this population. Allele misclassification was caused by poor amplification of the DNA in six samples and superimposition of glycosylated and nonglycosylated apo E isoforms on isoelectric focusing gels in five samples. We conclude that the assumption of genetic homogeneity among genotype classes is valid and that misclassification due to technical difficulties is more frequent than true discordancies.


Effect of cohort differences in smoking prevalence on models of lung cancer susceptibility.

Data on 337 lung cancer families were analyzed to determine if known cohort differences in parental cigarette consumption influence parameters from a segregation analysis. Previous results suggested that, after allowing for an individual's pack-years of tobacco exposure, Mendelian codominant inheritance of an allele that produced an earlier age of onset provided a good fit to the data. In the present study, the data were split into two groups of families: probands age 60 and over (born before WWI) and probands younger than age 60. This partition of the data by age of the proband was done to separate families in which there were parents who were less likely to smoke from those with parents more likely to smoke--predicated on the known increase of smoking prevalence after World War I. For the younger proband families (those with parents more likely to smoke), only Mendelian codominant inheritance adequately fit the data. The hypotheses of no major type, environmental transmission, and Mendelian dominant or recessive inheritance were rejected. In contrast to our earlier findings, the estimate of population susceptibility increased from 28% in the total data to 60% in this subset. In the older proband families (those with parents less likely to smoke), the no major type and environmental hypotheses were rejected; further, none of the Mendelian models could be distinguished. Our results demonstrate that cohort differences, probably in exposure to tobacco, can confound parameters of a segregation analysis, and suggest that the genetic component of lung cancer may be greater than previously estimated. It further suggests that susceptibility to lung cancer occurs as a function of susceptibility to the effects of tobacco smoking.


Elementary methods for the analysis of dichotomous outcomes in unselected samples of twins.

This paper presents an elementary statistical method for analyzing dichotomous outcomes in unselected samples of twin pairs using stratified estimators of the odds ratio. The methodology begins by first randomly designating one member of each twin pair as an "index" twin and the other member as the "co-twin." Stratifying on zygosity, odds ratios are used to measure the association between disease in the index twin and disease in the co-twin. From these zygosity-specific tables we calculate the Woolf-Haldane estimator of the common odds ratio (psi F, the weighted average of the zygosity-specific odds ratios), the Mantel-Haenszel test statistic (chi 2M-H) for the common odds ratio, and a test (chi 2G) for the difference in the zygosity-specific odds ratios. In this application, psi F provides an estimate of the familial association for disease and the accompanying chi 2M-H provides a test of the null hypothesis, psi F = 1 (i.e., there is no evidence for a familial influence on disease). The chi 2G is a test of the null hypothesis that psi MZ = psi DZ; a significant value for chi 2G suggests a genetic influence on disease (assuming that the observed odds ratios follow a pattern where psi MZ greater than psi DZ). A new test statistic (chi 2c) is proposed that incorporates the expectation that psi MZ = psi 2DZ under a purely additive genetic model with no common environmental effects. A significant value of chi c2 indicates that the different odds ratios across zygosity are partly due to common environmental influences. Conversely, a nonsignificant value of chi 2c is an indication that the zygosity-specific odds ratios are due solely to additive genetic effects and not to common environment. This basic approach is extended to examine the effects of measured indicators of the specific environment and the assessment of certain forms of gene by environment interaction. All of the methods are easily understood, highly flexible, readily computed using a hand calculator, and incorporate the inherent genetic information contained within twin samples.


Analysis of a gene conversion gradient at the HIS4 locus in Saccharomyces cerevisiae.

Heteroduplexes formed between genes on homologous chromosomes are intermediates in meiotic recombination. In the HIS4 gene of Saccharomyces cerevisiae, most mutant alleles at the 5' end of the gene have a higher rate of meiotic recombination (gene conversion) than mutant alleles at the 3' end of the gene. Such gradients are usually interpreted as indicating a higher frequency of heteroduplex formation at the high conversion end of the gene. We present evidence indicating that the gradient of conversion at HIS4 primarily reflects the direction of mismatch repair rather than the frequency of heteroduplex formation. We also identify a site located between the 5' end of HIS4 and the 3' end of BIK1 that stimulates heteroduplex formation at HIS4 and BIK1.


Isolation of Neurospora crassa A mating type mutants by repeat induced point (RIP) mutation.

In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.


Correlation between pairing initiation sites, recombination nodules and meiotic recombination in Sordaria macrospora.

The decrease of meiotic exchanges (crossing over and conversion) in two mutants of Sordaria macrospora correlated strongly with a reduction of chiasmata and of both types of "recombination nodules." Serial section reconstruction electron microscopy was used to compare the synapsis pattern of meiotic prophase I in wild type and mutants. First, synapsis occurred but the number of synaptonemal complex initiation sites was reduced in both mutants. Second, this reduction was accompanied by, or resulted in, modifications of the pattern of synapsis. Genetic and synaptonemal complex maps were compared in three regions along one chromosome arm divided into well marked intervals. Reciprocal exchange frequencies and number of recombination nodules correlated in wild type in the three analyzed intervals, but disparity was found between the location of recombination nodules and exchanges in the mutants. Despite the twofold exchange decrease, sections of the genome such as the short arm of chromosome 2 and telomere regions were sheltered from nodule decrease and from pairing modifications. This indicated a certain amount of diversity in the control of these features and suggested that exchange frequency was dependent not only on the amount of effective pairing but also on the localization of the pairing sites, as revealed by the synaptonemal complex progression in the mutants.


Molecular population genetics of an electrophoretically monomorphic protein in the alcohol dehydrogenase region of Drosophila pseudoobscura.

Nucleotide sequence data from the alcohol dehydrogenase (Adh) region of 18 isochromosomal strains of Drosophila pseudoobscura were used to determine whether the lack of amino acid polymorphism in ADH results from a low neutral mutation rate or a recent directional selection event. We estimated the neutral mutation parameter, 4Nmu, in synonymous sites for 17 subregions of Adh. The nucleotide diversity data were tested for departures from an equilibrium neutral model with two statistical tests. The Tajima test and the Hudson, Kreitman and Aguade test each failed to reject a neutral model. These results suggest that the ADH enzyme of D. pseudoobscura lacks amino acid polymorphisms because the neutral mutation rate of nonsynonymous sites is low. The neutral mutation parameter for synonymous sites is heterogeneous between domains of the Adh region. These data indicate that selective constrains on synonymous sites can vary between functional domains.


Differentiation of a male-specific muscle in Drosophila melanogaster does not require the sex-determining genes doublesex or intersex.

A pair of muscles span the fifth abdominal segment of male but not female Drosophila melanogaster adults. To establish whether genes involved in the development of other sexually dimorphic tissues controlled the differentiation of sex-specific muscles, flies mutant for five known sex-determining genes were examined for the occurrence of male-specific abdominal muscles. Female flies mutant for alleles of Sex-lethal, defective in sex determination, or null alleles of transformer or transformer-2 are converted into phenotypic males that formed male-specific abdominal muscles. Both male and female flies, when mutant for null alleles of doublesex, develop as nearly identical intersexes in other somatic characteristics. Male doublesex flies produced the male-specific muscles, whereas female doublesex flies lacked them. Female flies, even when they inappropriately expressed the male-specific form of doublesex mRNA, failed to produce the male-specific muscles. Therefore, the wild-type products of the genes Sex-lethal, transformer and transformer-2 act to prevent the differentiation of male-specific muscles in female flies. However, there is no role for the genes doublesex or intersex in either the generation of the male-specific muscles in males or their suppression in females.


Frequencies of mitochondrial DNA haplotypes in laboratory cage populations of the mosquito, Aedes albopictus.

A laboratory cage experiment was undertaken to study changes over time in the frequencies of two mitochondrial DNA (mtDNA) haplotypes in the mosquito, Aedes albopictus, under two conditions: bidirectionally compatible matings and unidirectionally incompatible matings. Frequencies were monitored for 10 generations in three replicate cages for each of the two conditions above. In cages with bidirectionally compatible strains, changes in haplotype frequencies were nondirectional and neither haplotype increased in frequency. Statistical analysis of relative proportions of the two haplotypes in each generation indicated that the magnitude of the observed fluctuations could be expected under an assumption of random genetic drift alone. In cages with unidirectionally incompatible matings, mtDNA of females that lay inviable eggs upon mating with males of another strain, decreased significantly in the F1 generation and was completely replaced in the F2 generation.


Variable rates of evolution among Drosophila opsin genes.

DNA sequences and chromosomal locations of four Drosophila pseudoobscura opsin genes were compared with those from Drosophila melanogaster, to determine factors that influence the evolution of multigene families. Although the opsin proteins perform the same primary functions, the comparisons reveal a wide range of evolutionary rates. Amino acid identities for the opsins range from 90% for Rh2 to more than 95% for Rh1 and Rh4. Variation in the rate of synonymous site substitution is especially striking: the major opsin, encoded by the Rh1 locus, differs at only 26.1% of synonymous sites between D. pseudoobscura and D. melanogaster, while the other opsin loci differ by as much as 39.2% at synonymous sites. Rh3 and Rh4 have similar levels of synonymous nucleotide substitution but significantly different amounts of amino acid replacement. This decoupling of nucleotide substitution and amino acid replacement suggests that different selective pressures are acting on these similar genes. There is significant heterogeneity in base composition and codon usage bias among the opsin genes in both species, but there are no consistent relationships between these factors and the rate of evolution of the opsins. In addition to exhibiting variation in evolutionary rates, the opsin loci in these species reveal rearrangements of chromosome elements.


Mitochondrial DNA length variation and heteroplasmy in populations of white sturgeon (Acipenser transmontanus).

Southern blot analysis was used to quantify the extent of mtDNA D-loop length variation in two populations of white sturgeon, Acipenser transmontanus. Over 42% of individuals were heteroplasmic for up to six different mtDNA length variants attributable to varying copy numbers of an 82-bp repeat sequence. Chi-square analyses revealed that the frequencies of length genotypes and the incidence of heteroplasmy were significantly different between Fraser and Columbia River sturgeon populations but not between restriction site haplotypes. Heteroplasmic fish have, on average, higher copy number than homoplasmic fish. Forty-five of 101 homoplasmic individuals carry only a single copy of the repeat, while none of the 73 heteroplasmic fish has the single repeat as the predominant variant. On the basis of differences in frequency distributions of copy number within and between fish, we suggest that (1) heteroplasmy is maintained by high recurrent mutation of multiple copy genomes, favoring increased copy number and (2) the mutation pressure toward higher copy number heteroplasmy is partially offset by selection to reduced genome size and segregation to the homoplasmic condition.


Traits that influence longevity in mice: a second look.

Analysis of genetic interactions in the F2 of an intercross of (C57BL/6 x DBA/2) F1J revealed influences of genetic factors on life span. Females lived longer than males. Dilute brown females died sooner than females of other colors. H-2b/H-2b males died sooner than H-2b/H-2d or H-2d/H-2d males, except that among dilute brown males those of typeH-2b/H-2d died sooner. Cluster analysis suggested that male and female genotypes each fall into two groups, with female dilute brown mice having shorter lives than other females, and male H-2b/H-2b mice except dilute brown and dilute brown H-2b/H-2d mice having shorter lives than other males. The association of heterozygosity with life span was clearer in females than in males, yet the longest-lived female genotype was homozygous H-2d/H-2d, of dominant Black phenotype at the Brown locus of chromosome 4, and homozygous dd at the Dilute locus of chromosome 9. The shortest-lived females were dilute brown H-2b/H-2b. The longest-lived and shortest-lived male genotypes were dilute brown H-2d/H-2d and dilute brown H-2b/H-2d, respectively. Although histological findings at postmortem differed between the sexes, there was no association of particular disorders with other genetic markers. The importance of H-2 in males was confirmed, but the allelic effects were perturbed, possibly by the absence of Sendai infection in this experiment. Overall our studies suggest that genetic influences on life span involve interactions between loci, and allelic interactions may change with viral infections or other environmental factors.


The polar-lethal Ovum mutant gene maps to the distal portion of mouse chromosome 11.

Genome imprinting is the process by which identical alleles at a particular locus may be rendered functionally different depending on the sex of the parent contributing the allele. While several mutations in imprinted genes have been defined, no variants in the regulatory system that gives rise to imprinting have been described. Here we report our genetic analysis of the behavior of the interstrain, polar, embryonic-lethal phenotype known as the "DDK syndrome." We have mapped the interstrain, polar-lethal region of the genome to the distal portion of mouse chromosome 11, near the Xmv-42 locus. We propose that the lethal phenotype is not caused by a standard mutation, but by aberrant imprinting of a gene within this region.


Simulation study of a multigene family, with special reference to the evolution of compensatory advantageous mutations.

We investigate the evolution of a multigene family incorporating the forces of drift, mutation, gene conversion, unequal crossing over and selection. The use of simulation studies is required due to the complexity of the model. Selection is modeled in two modes: positive selection as a function of the number of different beneficial alleles and negative selection against deleterious alleles. We assume that gene conversion is unbiased, and that all mutations are initially deleterious. Compensation between mutants creates beneficial and neutral alleles, and allowances are made for compensatory mutations either within or between the members of a multigene family. We find that gene conversion can enhance the rate of acquisition of compensatory advantageous mutations when genes are redundant.


Comparative effects of pollen and seed migration on the cytonuclear structure of plant populations. II. Paternal cytoplasmic inheritance.

We continue our study of the effects of pollen and seed migration on the cytonuclear structure of mixed-mating plant populations by analyzing two deterministic continent-island models under the critical assumption of paternal cytoplasmic inheritance. The major results of this study that contrast with our previous conclusions based on maternal cytoplasmic inheritance are (i) pollen gene flow can significantly affect the cytonuclear structure of the island population, and in particular can help to generate cytonuclear disequilibria that greatly exceed the magnitude of those that would be produced by seed migration or mixed mating alone; (ii) with simultaneous pollen and seed migration, nonzero cytonuclear disequilibria will be maintained not only when there is disequilibrium in the immigrant pollen or seeds, but also through a variety of intermigrant admixture effects when the two pools of immigrants differ appropriately in their cytonuclear compositions; (iii) either immigrant pollen or immigrant seeds can generate disequilibria de novo in populations with initially random cytonuclear associations, but pollen migration alone generally produces lower levels of disequilibrium than does comparable seed migration, especially at high levels of self-fertilization when the overall fraction of immigrant pollen is low; (iv) the equilibrium state of the island population will be influenced by the rate of pollen gene flow whenever there is either allelic disequilibrium in the immigrant pollen or simultaneous seed migration coupled with different cytoplasmic or nuclear allele frequencies in immigrant pollen and seeds or nonzero allelic disequilibrium in either immigrant pool. The estimation of pollen migration should therefore be facilitated with paternal cytoplasmic inheritance relative to the case of maternal cytoplasmic inheritance. These basic conclusions hold whether the population is censused as seeds or as adults, but with simultaneous pollen and seed migration, the relationship between census time and the ability to detect nonrandom cytonuclear associations is complex. When migration is through pollen alone, however, the cytonuclear structure of the island population is independent of the life stage censused.


Testing for equality of evolutionary rates.

A likelihood ratio test is presented for comparing rates of evolutionary change in the paths of descent leading to two species. The test is compared to previous relative rate tests based on variances of estimated numbers of base substitutions. The likelihood approach allows for different transversion and transition rates, and when these rates are actually different, the likelihood ratio test can be much more powerful than the variance-based tests. For single-parameter mutation models, however, the two tests have similar power. The tests are applied to a set of chloroplast sequences from several species of grasses, and additional indications of significantly different rates leading to barley were found with the likelihood ratio test.


Inferring selective history from multilocus frequency data: Wright meets the Hamiltonian.

We describe a method to study characteristics of the dynamics of multilocus population genetic models without specifying the form of selection a priori. Our approach consists of specifying initial and final genotypic frequencies (either completely or partially) and then determining the minimum time to go from the initial condition to the final condition according to a continuous time genetic model, with arbitrary constraints on the strength and possibly the form of selection. In analyzing a two-locus, two-allele model with this approach, we show that--so long as r is not much larger than s--substantial linkage disequilibrium can be generated from an initial state of linkage equilibrium in a few hundred generations. We also show that unless recombination is much larger than selection, there is only weak dependence on r of the minimum time to reach a specified state. Thus, similar strengths of selection can lead to similar levels of disequilibrium over a fixed time and a range of small recombination rates. This implies that, within the level of a single gene, selection cannot in general be assumed to lead to any particular relationship between recombination rate and levels of disequilibrium. We indicate a number of other ways in which our method can be useful in asking theoretical questions and in interpreting data.


A colony color assay for Saccharomyces cerevisiae mutants defective in kinetochore structure and function.

We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae). The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter. Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct. A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the "pseudo-operator" led to the identification of mutations (blu) in three complementation groups. The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro. The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes. Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins.


Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.


Possible cross-regulation of phosphate and sulfate metabolism in Saccharomyces cerevisiae.

CP1 (encoded by the gene CEP1) is a sequence-specific DNA binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 exhibit defects in growth, chromosome segregation and methionine biosynthesis. A YEp24-based yeast genomic library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4. PHO4c, pho80 and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy. The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was PHO2-dependent. Spontaneously arising extragenic suppressors of cep1 methionine auxotrophy were also isolated; approximately one-third of them were alleles of pho80. While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low inorganic phosphate growth deficiency of a pho84 mutant. The results may suggest that phosphate and sulfate metabolism are cross-regulated.


MAK10, a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae, has T cell receptor alpha-subunit motifs.

The MAK10 gene is necessary for the propagation of the L-A dsRNA virus of the yeast Saccharomyces cerevisiae. We have isolated MAK10 from selected phage lambda genomic DNA clones that map near MAK10. This gene encodes a 733-amino acid protein with several regions of similarity to T cell receptor alpha-subunit V (variable) regions. We show that MAK10 is essential for optimal growth on nonfermentable carbon sources independent of its effect on L-A. Although loss of L-A by mak10-1 mutants is partially suppressed by loss of the mitochondrial genome, no such suppression of a mak10::URA3 mutation was observed. Using MAK10-lacZ fusions we show that MAK10 is expressed at a very low level and that it is glucose repressed. The highest levels of expression were seen in tup1 and cyc8 mutants, known to be defective in glucose repression. These results suggest that the mitochondrial genome and L-A dsRNA compete for the MAK10 protein.


An examination of adaptive reversion in Saccharomyces cerevisiae.

Reversion to Lys+ prototrophy in a haploid yeast strain containing a defined lys2 frameshift mutation has been examined. When cells were plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. An examination of the distribution of the numbers of early appearing Lys+ colonies from independent cultures suggests that the mutations to prototrophy occurred randomly during nonselective growth. In contrast, an examination of the distribution of late appearing Lys+ colonies indicates that the underlying reversion events occurred after selective plating. No accumulation of Lys+ revertants occurred when cells were starved for tryptophan, leucine or both lysine and tryptophan prior to plating selectively for Lys+ revertants. These results indicate that mutations accumulate more frequently when they confer a selective advantage, and are thus consistent with the occurrence of adaptive mutations in yeast.


Cyclin E/cdk2 and cyclin A/cdk2 kinases associate with p107 and E2F in a temporally distinct manner.

Cyclin E is classified as a putative G1 cyclin on the basis of its cyclic pattern of mRNA expression, with maximal levels being detected near the G1/S boundary. We report here that cyclin E is found associated with the transcription factor E2F in a temporally regulated fashion. E2F is known to be a critical transcription factor for the expression of some S phase-specific proteins and is thought to be important for a series of others. Antisera specific for cyclin E were raised and used to demonstrate an association between cyclin E and E2F. This cyclin E/E2F complex was seen in a variety of human cell lines from various tissues, but its appearance was detected primarily during the G1 phase of the cell cycle. The cyclin E/E2F association decreased as cells entered S phase, just as the association of E2F with cyclin A became detectable. We characterized the cyclin E-E2F complex further to show that both the cyclin-dependent kinase-2 (cdk2) and p107 were present. Therefore, the p107/E2F complex is associated with two different cdk2 kinase complexes--one containing cyclin A and the other containing cyclin E--and the appearance of these complexes is temporally regulated during the cell cycle. The presence of cyclin E/E2F complexes in the G1 phase suggests a role for cyclin E in the control of genes required for the G1-to-S transition.


Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53.

Wild-type p53 protein was shown to bind specifically to DNA sequences within SV40 (Bargonetti et al. 1991), the human ribosomal gene cluster (RGC) (Kern et al. 1991a), and the murine muscle creatine kinase gene (MCK) (Zambetti et al. 1992). However, a direct comparison of these three sites was not performed. Here we demonstrate, by filter binding and gel mobility-shift assays, that wild-type p53 binds with similar affinities to MCK and RGC sites but less tightly to the SV40 site. We examined the effects of two candidate regulators of p53 function, SV40 large T antigen and oncogenic mutant p53, on the binding of wild-type p53 to RGC DNA. We show that wild-type T antigen prevents p53 from binding to the RGC site under all conditions tested. Moreover, two temperature-sensitive mutant SV40 T antigens, which fail to transform cells at the nonpermissive temperature, prevent p53 from binding to the RGC site at the permissive, but not at the restrictive, temperature. The ability of complexes containing wild-type p53 and tumor-derived mutant p53 proteins to bind to RGC DNA varies according to the position of the mutation. Complexes containing wild-type and either his175 or his273 mutant p53 proteins are completely unable to bind to the RGC DNA sequence. Interestingly, a complex containing wild-type p53 and the trp248 mutant p53 characteristic of Li-Fraumeni syndrome patients displays nearly wild-type levels of binding. Perhaps this mutant allele can be tolerated in these individuals because the wild-type mutant p53 complex maintains the ability to bind to DNA. Our data indicate that the oncogenic potential of both T antigen and some mutant p53 proteins is the result of their ability to block binding of wild-type p53 to DNA.


Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure.

A temperature-sensitive lethal mutation in Saccharomyces cerevisiae, prp20-1, causes defects in several different steps in mRNA metabolism, including mRNA 3'-end formation, transcription initiation, and mRNA transport. Previous work has demonstrated that prp20 mutants are defective in actin pre-mRNA splicing. PRP20 is related, both in structure and function, to the RCC1 gene of mammals and the PIM1 gene of Schizosaccharomyces pombe, both of which appear to regulate entry into mitosis and chromosome condensation. In this report we demonstrate that, after a shift of prp20 mutants to the restrictive temperature, transcripts of several genes (CUP1, CYH2, and GAL10) are produced that extend 1-10 kb beyond their normal polyadenylation sites. The failure in 3'-end formation occurs within 1-2 min of the temperature shift. Transcription initiation also is disrupted, in that initiation sites upstream of the normal cap site are used. mRNA transport from nucleus to cytoplasm also is perturbed: In situ hybridization using an oligo(dT) probe demonstrates accumulation of poly(A) in the nucleus, consistent with the accumulation of longer bulk poly(A) (up to approximately 90-100 nucleotides) and with a failure to transport newly synthesized RNA to the cytoplasm. We demonstrate that prp20 and rna1 mutants are very similar, if not identical, with respect to each of these biochemical phenotypes. In light of the putative role of PRP20 in mitotic control, our results suggest a common step in that process and multiple steps in mRNA synthesis and maturation. We speculate that the perturbations in mRNA processing are the result of effects on the chromatin-nascent RNP-transcription complex or misregulation of a cell cycle component that modifies multiple mRNA-processing activities.


Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex.

Many short-lived mRNAs, including those encoding lymphokines, cytokines, and proto-oncogenes, contain an AU-rich sequence in their 3'-untranslated regions. These AU domains and, more specifically, AUUUA motifs within them, are widely thought to mediate the extreme instability of the corresponding mRNAs. This is most clearly true for granulocyte monocyte colony stimulating factor (GM-CSF) mRNA whose AUUUA motifs are conserved phylogenetically and whose presence in an otherwise stable beta-globin mRNA results in a 50-fold decrease in accumulated mRNA level. We show that RNA instability conferred by the GM-CSF AU motif requires the mRNA to be actively translated and the AU motif to be within the 3'-untranslated region. By analysis of the sedimentation characteristics, we identify a large (> 20S), divalent cation-independent complex found only on unstable RNAs. Like instability, complex formation (1) is dependent on translation of the RNA, (2) requires intact AUUUA motifs, and (3) is blocked by ribosome translocation across the AU-rich motif. We propose that RNA instability mediated by the AU motif is achieved through translation-dependent assembly of this large mRNA-destabilizing complex.


A yeast TFIIB-related factor involved in RNA polymerase III transcription.

A suppressor gene was identified, which in high copy number rescues a temperature-sensitive mutation in yeast TATA-binding protein (TBP). Suppression was allele specific because the suppressor did not rescue the temperature-sensitive phenotype of another TBP mutant. This suppressor gene encodes a 596-amino-acid protein of which the amino-terminal half is homologous to the Pol II-specific factor TFIIB. Disruption of this gene, termed BRF1, showed it to be essential for growth of yeast. Deletion of sequences at either the amino or carboxyl terminus of BRF1 gave both temperature- and cold-sensitive phenotypes. These temperature- and cold-sensitive strains were used to prepare extracts deficient in BRF1 activity and were tested for transcriptional activity by RNA polymerases I, II, and III in vitro. BRF1-deficient extracts are defective in Pol III transcription and can be reconstituted for Pol III transcription by the addition of recombinant BRF1. Western analysis shows that BRF1 is present in TFIIIB but not the TFIIIC fraction, suggesting that it is a component of TFIIIB. We propose that BRF1 plays a role in Pol III initiation analogous to the role played by TFIIB for Pol II in its interaction with TBP and polymerase. The identification of a Pol III-specific TFIIB-like factor extends the previously noted similarity of transcriptional initiation by the three nuclear polymerases.


Multiple domains of the RNA polymerase I activator hUBF interact with the TATA-binding protein complex hSL1 to mediate transcription.

Recent evidence suggests that transcription initiation by all three eukaryotic RNA polymerases involves a complex of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, we map the functional domains of the nucleolar HMG box protein hUBF, which binds to the human rRNA promoter and stimulates transcription by RNA polymerase I through cooperative interactions with a distinct TBP-TAF complex, hSL1. DNase I footprint analysis of mutant hUBF proteins and of a synthetic peptide of 84 amino acids reveals that HMG box 1 is necessary and sufficient for DNA sequence specificity, whereas other HMG boxes and the amino terminus modulate the binding efficiency. hUBF contains multiple activation domains that include the acidic carboxyl terminus and three HMG boxes. HMG boxes 3 and 4 and the acidic tail contribute significantly to an extended footprinting pattern in the presence of hSL1, suggestive of specific protein-protein interactions. Moreover, the inability of xUBF from Xenopus laevis to form an initiation complex with hSL1 can be overcome by hybrid proteins containing human HMG box 4 and the acidic carboxyl terminus. These results strongly suggest an important role of transcription activation domains of hUBF in mediating interactions with the TBP-TAF complex hSL1.


Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter.

Transcription factor IID (TFIID) binds to TATA boxes, nucleating the assembly of initiation complexes containing several general transcription factors and RNA polymerase II. Recently, TFIID was shown to be a multisubunit complex containing a TATA box-binding polypeptide (TBP) and several tightly associated polypeptides (TAFs), which are required for transcriptional stimulation by activator proteins. Here, we report the development of a human cell line expressing an epitope-tagged TBP and the immunopurification of a native, high-molecular-weight form of TFIID that supports transcriptional stimulation by several different classes of activation domains. Recovery of basal and activated TFIID transcriptional specific activity was close to approximately 100%. Electrophoretic mobility-shift analysis demonstrated a single major DNA-protein complex. This holo-TFIID contains TAFs of approximately 250, 125, 95, 78, and 50 kD and sediments at 17S. Holo-TFIID produced an extended footprint over the adenovirus major late promoter TATA box and initiator sequence and supported transcriptional activation from a promoter lacking a TATA box. These results lead us to hypothesize that a single multisubunit TFIID protein supports transcriptional stimulation by diverse activation domains and from a TATA-less promoter.


Yeast site-specific ribonucleoprotein endoribonuclease MRP contains an RNA component homologous to mammalian RNase MRP RNA and essential for cell viability.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA sequence complementary to mammalian mitochondrial origins of replication in a manner consistent with a role in primer RNA metabolism. The same activity in the yeast Saccharomyces cerevisiae has recently been identified; it cleaves an RNA substrate complementary to a yeast mitochondrial origin of replication at an exact site of linkage of RNA to DNA. We have purified this yeast enzyme further and detect a single, novel RNA of 340 nucleotides associated with the enzymatic activity. The single-copy nuclear gene for this RNA was sequenced and mapped to the right arm of chromosome XIV. The identity of the clone, as encoding the RNA copurifying with enzymatic activity, was confirmed by a match to the directly determined sequence of the RNA. The gene sequence also identified a 340-nucleotide RNA in total yeast RNA and in purified RNase MRP enzyme preparations. Inspection of the sequence of the yeast RNA revealed homologies to the RNA component of mouse RNase MRP, 49% overall with specific regions of much greater similarity. The flanking regions of the gene showed characteristics of an RNA polymerase II transcription unit, including a TATAAA box and a 7/8 match to the yeast cell cycle box UAS. The RNase MRP RNA gene was deleted by insertional replacement and found to be essential for cellular viability, indicating a critical nuclear role for RNase MRP.


Protein components specifically associated with prespliceosome and spliceosome complexes.

We have carried out a systematic analysis of the protein composition of highly purified mammalian spliceosomes. We show that > 30 distinct proteins, including 20 previously unidentified components [designated spliceosome-associated proteins (SAPs)], are specifically associated with the spliceosome in a salt-resistant complex. In contrast to these spliceosome-specific proteins, we show that hnRNP proteins are not tightly associated with purified prespliceosome and spliceosome complexes. The splicing factor U2AF65, U1 snRNP-specific proteins, and several SAPs are present in the earliest prespliceosome complex (E). A set of 10 proteins is then added to the first ATP-dependent prespliceosome complex (A), and concomitantly, a significant decrease in the level of U2AF65 is observed. The fully assembled spliceosome is formed by the addition of 12 proteins in a reaction that requires ATP and both the 5' and 3' splice sites.


The Escherichia coli rna gene encoding RNase I: sequence and unusual promoter structure.

A clone containing the Escherichia coli rna gene encoding the nonspecific endoribonuclease, RNase I, was isolated and sequenced. The sequence of the 1070-nucleotide (nt) fragment agreed completely with that of a rna clone recently reported by Meador and Kennell [Gene 95 (1990) 1-7]. The transcription start point (tsp) of rna was identified using primer extension analysis, and its promoter sequence was established by comparison of RNase I expression levels in various deletion mutants. Our results indicate that the rna promoter is highly unusual. Its -35 region shows a poor match to the consensus sequence, and moreover, it is located within a stem-loop structure that apparently is a Rho-independent transcription termination site for an upstream gene.


Sequence analysis of a DNA fragment from Buchnera aphidicola (an endosymbiont of aphids) containing genes homologous to dnaG, rpoD, cysE, and secB.

The aphid, Schizaphis graminum, contains a prokaryotic, obligately intracellular endosymbiont, Buchnera aphidicola, which is necessary for the survival of the host. A recent study of Bu. aphidicola 16S rRNA has indicated that it is a member of the gamma-3 subdivision of the eubacterial class, Proteobacteria, which includes Escherichia coli. In order to further characterize the endosymbiont and establish its similarity to free-living eubacteria and/or organelles, we have cloned and sequenced a 4534-bp DNA fragment containing dnaG-rpoD-cysE-secB. The deduced amino acid (aa) sequence identity to the homologous E. coli proteins ranged from 47 to 80%. The close proximity of the pair, dnaG-rpoD, to the pair, cysE-secB, on the Bu. aphidicola DNA, differed from E. coli, in which these two pairs of genes are 14 min apart on the bacterial chromosome. The results of past physiological studies of the endosymbiont were consistent with the presence and function of DNA primase (DnaG), sigma factor (RpoD) and components of the secretory system (SecB). Comparison of the deduced aa sequence of Bu. aphidicola CysE (serine acetyltransferase, a key allosterically regulated enzyme in cysteine biosynthesis) with the E. coli wild-type enzyme and a mutant defective in feedback inhibition suggested that the endosymbiont CysE may not be regulated. By analogy with E. coli, the lack of feedback inhibition may lead to overproduction of cysteine by the endosymbiont. The results of this and previous investigations indicate that Bu. aphidicola has many of the properties of free-living bacteria and not of organelles.


Production of an enzymatically inactive analog of phospholipase D from Corynebacterium pseudotuberculosis.

The gene pld, encoding the phospholipase D (PLD) of Corynebacterium pseudotuberculosis, was mutagenized using formic acid and then expressed in Escherichia coli. Mutagenesis was targeted at the coding region of pld, so as to produce only one or a limited number of point mutations. Transformants were screened for the enzymatic and immunological properties of their PLD products. One clone was found to produce a protein which was enzymatically inactive, but which was comparable to the wild-type PLD in size and antigenicity. The sequence of the pld mutant revealed a single base change. As a consequence, the codon for His20 was converted to Tyr. These results suggest that His20 forms part of the active site of the PLD molecule. If this protein is immunogenic in sheep, it would form the basis of a genetically inactivated vaccine.


Isolation and complete sequence of the purL gene encoding FGAM synthase II in Lactobacillus casei.

The purL gene from Lactobacillus casei, encoding phosphoribosylformylglycinamidine synthase II involved in the de novo synthesis of purines, was cloned and sequenced. The putative purL product of 741 amino acids (M(r) of 79,575) shows 25% and 53% identity to the homologous enzymes from Escherichia coli and Bacillus subtilis, respectively. In addition, partial sequences of two other pur genes (purQ and purF) and a possible third gene (purC) were obtained. All these genes are organized in an operon similar to that of B. subtilis. In contrast, the corresponding genes from E. coli and Salmonella typhimurium are scattered through the genome.


Genetic transformation of a halophilic archaebacterium with a gas vesicle gene cluster restores its ability to float.

The halophilic archaebacterium, Halobacterium halobium, and many other aquatic bacteria synthesize gas-filled vesicles for flotation. We recently identified a cluster of 13 genes (gvpMLKJIHGFEDACN) on a 200-kb H. halobium plasmid, pNRC100, involved in gas vesicle synthesis. We have cloned and reconstructed the gvp gene cluster on an H. halobium-E. coli shuttle plasmid. Transformation of H. halobium Vac- mutants lacking the entire gas vesicle gene region with the gvp gene cluster results in restoration of their ability to float. These results open the way toward further genetic analysis of gas vesicle gene functions and directed flotation of other microorganisms with potential biotechnological applications.


pYLZ vectors: Saccharomyces cerevisiae/Escherichia coli shuttle plasmids to analyze yeast promoters.

Two yeast/Escherichia coli shuttle vectors have been constructed to analyze promoter structures in Saccharomyces cerevisiae: the multicopy vector, pYLZ-2, and the centromere-based vector, pYLZ-6. Both plasmids contain the coding region of lacZ from E. coli lacking the N-terminal eight amino acids. The truncated reporter gene is preceded by a short polylinker (MCS) suitable for the insertion of promoter fragments. The vectors allow for the study of expression from complete promoters containing UAS and TATA elements, transcriptional start point(s) and the original context of the ATG start codon of a yeast gene. A yeast terminator fragment has been inserted 3' of the lacZ coding region. It contains the transcription termination region of the convergently transcribed yeast genes, GCY1 and PFY1, together with sequences corresponding to the mapped 3'-ends of the respective mRNAs. As an example, reporter activity was measured with promoter fragments from three yeast genes (GCY1, PFY1 and LEO1). The results demonstrate the efficiency of the plasmids for studying constitutive and regulated transcription, both at high and low levels of expression.


Sequence of the subtilisin-encoding gene from an antarctic psychrotroph Bacillus TA41.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site.


Sequence encoding ribosomal protein L33 of Lactococcus lactis.

A cloned fragment from Lactococcus lactis chromosome encoding the L33 ribosomal protein was sequenced. Two incomplete open reading frames (ORFs) were also found: the upstream ORF shows similarity to the tetracycline-resistance protein (Tet) of Bacillus stearothermophilus, and the downstream ORF shows homology to a protein of Bacillus subtilis participating in sporulation (SpoVE), and to proteins of Escherichia coli involved in cell division (FtsW) and the maintenance of cell shape (RodA).


Cloning and sequencing of the gene encoding a ribonuclease from Streptomyces aureofaciens CCM3239.

A ribonuclease-encoding gene (rnaSa3) from Streptomyces aureofaciens CCM3239 has been isolated and sequenced. The deduced amino acid sequence shows 77% homology with RNase Sa from S. aureofaciens.


Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp.

New multiple cloning sites (MCS), which facilitate the subcloning of G+C-rich DNA, were added to pUC18, M13mp18, pVE616 (a pBR322-derived insertion vector), and the low-copy-number Streptomyces vector, pIJ922. The MCS in these vectors contain sites found infrequently in Streptomyces DNA, facilitating the exchange of subclones between the vectors. The MCS added to M13mp18 and pUC18 was also designed to generate nested deletions within subcloned fragments.


Sequence of the 5-aminolevulinic acid dehydratase-encoding gene from the hyperthermophilic methanogen, Methanothermus sociabilis.

We report on the sequence of the Methanothermus sociabilis aladh gene, which encodes the 5'-aminolevulinic acid dehydratase. The identity of the enzyme was determined by sequence comparison and by expression in Escherichia coli.


Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum.

The sequence of a 5633-bp EcoRI-PvuII DNA fragment from Clostridium cellulolyticum was determined. This fragment contains two complete endo-beta-1,4-glucanase-encoding genes, designated celCCC and celCCG. These two genes are flanked by two other partial open reading frames (ORF1 and celCCE) that probably encode two cellulases or related enzymes. The celCCC and celCCG genes appear to be present in a polycistronic transcriptional unit. Northern blot hybridisations with intragenic probes derived from celCCC and celCCG gave similar patterns. Two transcripts of about 5 and 6 kb were identified. The celCCC and celCCG ORFs extend over 1380 bp and 2175 bp, respectively. They are separated by only 87 nt. A typical signal sequence is present at the N terminus of the deduced polypeptides. The mature CelCCC and CelCCG proteins have M(r)s 47,201 and 76,101, respectively. Comparisons between their amino acid (aa) sequences and other known cellulase sequences revealed that: first, they both contain the repeated 24-aa sequence characteristic of clostridial beta-glycanases, secondly, the N-terminal catalytic domains of CelCCC and CelCCG can be classified into the D and E2 families, respectively, and thirdly, the largest CelCCG contains an additional internal domain which is very similar to that of the Bacillus-type cellulose-binding domain (CBD). The ORF1-C-terminal-encoded sequence also contains the clostridial 24-aa repeat. The CelCCE N-terminus consists of a typical signal sequence followed by a 168-aa domain homologous to the N-terminal repeated domain of Cellulomonas fimi CenC. This domain is connected to an incomplete catalytic domain of family E1 by a Pro-rich junction linker.