Restriction fragment length polymorphisms detected in N-ras-related sequences of rats and their linkage analyses

Novel restriction fragment length polymorphisms (RFLPs) in inbred rats were revealed with the human N-ras gene as probe. Three fragments hybridizing to the probe were detected by Southern blot hybridization under highly stringent conditions, and one of the fragments showed variation in inbred rat strains. Furthermore, on hybridization under low-stringency conditions, an additional fragment hybridizing to the probe was observed, and this fragment also showed interstrain variation. These two variant fragments showed different distributions in 27 inbred rat strains and segregated in backcross progeny as codominant alleles of independent single autosomal loci. Therefore, the loci for these RFLPs were named Nras-1 and Nras-2, respectively. Analyses of linkages between the RFLPs and 11 other loci revealed that the Nras-2 locus was closely linked to the c locus (3.7 +/- 2.6%), which belongs to rat linkage group I.     

The oxyvanadium constellation in transition-state-analogue complexes of phosphoglucomutase and ribonuclease. Structural deductions from electron-transfer spectra

The absorbance peak in the near ultraviolet electron-transfer spectrum of the oxyvanadium constellation in the "transition-state-analogue complexes" obtained by treating the dephospho form of phosphoglucomutase with inorganic vanadate in the presence of either glucose 1-phosphate or glucose 6-phosphate, as described in an accompanying paper [Ray, W. J., Jr., Burgner, J. W., II, & Post, C. B. (1990) Biochemistry (second of four papers in this issue)], is centered at a wavelength of 312 nm. The position of this peak amounts to a change in oscillator frequency of about -5000 cm-1 relative to that of tetrahedral VO4(3-). To provide a rationale for this spectral change, the near ultraviolet spectra of the di- and monoanions of inorganic vanadate and a number of derivatives of these anions are compared with that of vanadium (V) in the enzymic complexes, in terms of both what is observed experimentally and what is expected from crystal field theory. Comparisons in water and in largely anhydrous solvents show that water is not an essential element in the coordination sphere of inorganic vanadate or its mono- or diesters and hence that the coordination number of V(V) in such compounds likely is four. These comparisons also show that loss of solvating water from a 4-coordinate vanadate on binding cannot provide a rationale for the spectra of the enzymic complexes. Other comparisons show that neither the binding of metal ions nor protonation nor the binding of vanadate at a site with an unusually high or an unusually low dielectric constant can provide such a rationale. Further comparisons with vanadates known to be pentacoordinate strongly suggest that the coordination number of V(V) in the transition-state-analogue complexes of phosphoglucomutase does not exceed four. In fact, from the standpoint of crystal field theory the marked red shift observed in the electron-transfer absorbance spectrum of the oxyvanadium constellation in these complexes is more reasonably interpreted in terms of a decreased coordination at vanadium (V), viz., in terms of a weakened bonding between vanadium and one or more of its coordinating oxygens. This decreased coordination could be produced by a physical stretching of the vanadate ester linkage. By contrast, the near ultraviolet spectrum of the transition-state-analogue complex that ribonuclease forms with an adduct of uridine and vanadate [Lindquist, R. N., Lynn, J. L., & Lienhard, G. E. (1973) J. Am. Chem. Soc. 95, 8762] is similar to spectra of pentacoordinate model compounds of vanadium(V).(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of a vanadate-based transition-state-analogue complex of phosphoglucomutase by kinetic and equilibrium binding studies. Mechanistic implications

The inhibitor complex produced by the binding of alpha-D-glucose 1-phosphate 6-vanadate to the dephospho form of muscle phosphoglucomutase exhibits an unusually small dissociation constant: about 15 fM for the Mg2+ enzyme at pH 7.4, when calculated in terms of the tetraanion. Such tight binding suggests that the enzyme/vanadate/glucose phosphate complex mimics a state that at least approaches the transition state for (PO3-) transfer in the normal enzymic reaction. This hypothesis also is supported by the observation that replacement of Mg2+, the normal metal ion activator, by Li+, a poor activator, substantially reduces the binding constant for the glucose phosphate/vanadate mixed diester. Other indicators that support this hypothesis are described. One is the derived equilibrium constant for replacement of a PO4(2-) group in bound glucose bisphosphate by VO4(2-): 3 x 10(6) when the replaced group is the phosphate at the (PO3-) transfer site of the Mg2+ enzyme--in contrast to about 10 for the same replacement (of PO4(2-) by VO4(2-)) in an aqueous solution of a phosphate ester. Another is the greatly decreased rate at which Mg2+ dissociates from the glucose phosphate/vanadate complex of the enzyme, relative to the rate at which it dissociates from the corresponding bisphosphate complex (rate ratio less than or equal to 3 x 10(-4)), presumably because Mg2+ binds more tightly to the glucose phosphate/vanadate complex than to the corresponding bisphosphate complex. This apparent increase in Mg2+ binding occurs in spite of what appears to be a reduced charge density at the bound vanadate grouping, relative to the bound phosphate grouping, and in spite of the somewhat weaker binding of Mg2+ by dianionic vanadate than by the phosphate dianion. Although a direct assessment of the binding constant for Mg2+ was not possible, the equilibrium constant for Mg2+/Li+ exchange could be evaluated for the complexes of dephospho enzyme with glucose bisphosphate or glucose 1-phosphate 6-vanadate. The results suggest that the glucose phosphate/vanadate complex of the Mg2+ enzyme mimics a state about halfway between the ground state and the transition state for (PO3-) transfer. This estimate also is in accord with the binding of glucose phosphate/vanadate relative to that expected for transition-state binding of glucose bisphosphate. A possible scenario for the (PO3-) transfer catalyzed by the Mg2+ form of phosphoglucomutase is discussed, on the basis of these observations, together with possible reasons why the bound vanadate group appears to mimic an intermediate state for (PO3-) transfer rather than the ground state for phosphate binding.     

Polymorphisms detected in actin-related sequences of rats (Rattus norvegicus)

Southern blot hybridization of EcoRI digests of DNAs from 13 rat strains using human cardiac actin gene as a probe revealed polymorphisms in actin-related sequences of rats. EcoRI fragments of 11 kb, 7 kb, 6 kb, 5 kb, 4.5 kb and 4 kb detected in several strains were absent in the remaining strains. The presence of these fragments was suggested to be due to presence of extra sequences homologous to the actin genes, such as processed pseudogenes, in the particular strains. The 13 strains were assigned to each of 7 specific patterns of the polymorphic EcoRI fragments. It was concluded that the polymorphisms of actin-related sequences should be useful for genetic monitoring of laboratory rats.     

Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.     

A universal primer mixture for sequence determination at the 3′ ends of cDNAs

We have devised a universal primer which can be used to sequence the 3′-ends of cloned cDNAs containing a polyA tail. The primer consists of an equimolar mixture of three primers: 20 T nucleotides followed by either an A, C, or G nucleotide (5′→3′). With this primer mixture and the dideoxynucleotide chain termination method, we determined the 3′-terminal sequence of human β-actin cDNA in an Okayama-Berg vector, in four parallel sets of reactions containing either a single primer (T₂₀G, T₂₀C, or T₂₀A) or an equimolar mixture of all three primers. Priming with both T₂₀A and the triple mixture gave clearly readable results that agree with the known sequence of the human β-actin gene, and we have applied this method successfully to several other cDNAs in the Okayama-Berg expression vector. Use of this universal primer mixture facilitates determination of sequences at the 3′-ends of cDNAs while by-passing the polyA tail region.     

Developmental Changes in the Bombyxin-and Insulin-like Immunoreactive Neurosecretory System in the Wax Moth, Galleria mellonella

Four medial neurosecretory cells (MNC) and 4 lateral neurosecretory cells (LNC) in each brain hemisphere, and one pair of cells in each thoracic ganglion (TG) of Galleria larva react with antibodies against bombyxin and insulin. Material secreted from the MNC and LNC is released mainly in the corpora allata, and that from the TG through the ventral median nerves. Intrinsic secretory cells of the corpora cardiaca (CC) also contain bombyxin-like, but not insulin-like material. The immunoreactivities all disappear during molts and reappear with resumption of feeding. In the MNC and TG they reappear for less than a day, but in cells of the CC immunoreactivity reappears for the whole feeding period. Before pupation, the LNC become temporarily immunopositive towards the end of feeding period, and the MNC and TG during the wandering period, i.e. at the time of prothoracic gland stimulation. Immunoreactivity disappears during the pupal molt. In pupae it is present in the 4 pairs of MNC and 1–2 pairs of LNC 12–48 hr after ecdysis, and in cells of the CC from 12 hr after ecdysis until the end of the pupal instar. In adult, immunoreactivity is restricted to 2 pairs of the LNC and to CC cells.     

Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol

The influences of buffers and iron chelators on the rate of autoxidation of Fe²⁺ were examined in the pH range 6.0–7.4. The catalysis by Fe²⁺ and Fe³⁺ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe²⁺, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30°C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe³⁺ over Fe²⁺, Fe²⁺ autoxidizes rapidly in the pH range 6.0–7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe²⁺ stabilize both Fe²⁺ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe³⁺ in non-chelating buffers or in the presence of strong chelators of Fe²⁺, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe²⁺ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe²⁺ and Fe³⁺. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe²⁺ or both dithiothreitol and iron.     

Homozygous Robertsonian translocations in a fetus with 44 chromosomes

Summary We report the unique finding of a human fetus with 44 chromosomes with homozygous 14;21 translocations. This fetus appeared phenotypically normal but the long-term neurodevelopmental outcome had this pregnancy continued could not be predicted. We speculate one 14;21 translocation was inherited from her father and one arose de novo being maternal in origin. A previous sibling with psychomotor retardation has an abnormal chromosome complement of 45,XX,dup(7)(q21pter), t(14;21)(p11;q11). The mother's underlying disease, systemic lupus erythematosis (SLE), and her prior chemotherapy may have contributed to the appearance of these chromosome aberrations. It is interesting that although 14;21 translocations are among the commonest structural chromosome rearrangements in man, there are no previous reports in newborn surveys of a child with 44 chromosomes resulting from the mating of two identical Robertsonian translocation carrier parents.     

Depletion of epinephrine in rat hypothalamus by a dopamine agonist,pergolide

The i.p. injection of pergolide mesylate, a dopamine agonist, at doses of 0.3–0.6 mg/kg led to a decrease in epinephrine concentration in rat hypothalamus. After a 0.6 mg/kg dose of pergolide mesylate, epinephrine concentration in hypothalamus decreased within 2 hr, reached a minimum concentration at about 8 hrs, and then returned toward control values. Norepinephrine N-methyltransferase activity was not decreased after pergolide injection in vivo nor was it inhibited by pergolide added in vitro at concentrations as high as 10^–3 M. Higher i.p. doses of less potent dopamine agonists, apomorphine (10 mg/kg) and lergotrile (3 mg/kg), also decreased epinephrine concentration in hypothalamus. The pergolideinduced decrease in hypothalamic epinephrine concentration was prevented by pretreatment with haloperidol or spiperone., antagonists of dopamine receptors. Activation of dopamine receptors appears to result in a decrease in epinephrine concentration in rat brain, possibly due to, enhanced release of epinephrine.