Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2.

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.

Purification and characterization of type 1 protein phosphatase from Saccharomyces cerevisiae: effect of the R73C mutation.

Type 1 protein phosphatase encoded by the GLC7 gene was purified from Saccharomyces cerevisiae as a 1:1 complex with mammalian inhibitor 2 fused to glutathione S-transferase. The complex was inactive and required treatment with Co2+ and trypsin for maximal activity. The specific activity toward phosphorylase a was about 1.8 units/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 7.3, 81, and 0.30 nM, respectively. The complex could be activated by glycogen synthase kinase-3 in the presence of Mg2+ and ATP to 20% of the activity seen with Co2+ and trypsin. Thus, the catalytic properties of the yeast type 1 phosphatase are similar to those of the mammalian protein phosphatase 1. The R73C mutant phosphatase from the glycogen-deficient strain, glc7-1, purified as a 1:1 complex with the inhibitor 2 fusion, had a specific activity toward phosphorylase a of 0.9 unit/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 13. 1, 113, and 0.37 nM, respectively. The R73C mutation slightly decreases the specific activity and sensitivity to inhibitors, suggesting that changes in biochemical properties may affect glycogen levels. However, the modest changes are consistent with our previous proposal (E. M. Reimann et al., 1993, Adv. Protein Phosphatases 7,173-182) and with the results of Stuart et al. (1994, Mol. Cell. Biol. 14, 896-905) that the mutation may selectively alter the interaction of Glc7p with regulatory proteins.

Evidence for the importance of hydrophobic residues in the interactions between the cAMP-dependent protein kinase catalytic subunit and the protein kinase inhibitors.

The protein kinase inhibitors (PKIs) are potent inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase. In this study, the interaction between Phe10 of PKI and the C subunit residues Tyr235 and Phe239 was investigated using site-directed mutagenesis. Previous peptide studies as well as the crystal structure suggested that these residues may play a key role in C-PKI binding. The C subunit codons for Tyr235 and Phe239 were changed singly and in combination to serine codons. The mutated C alpha proteins were overexpressed in Escherichia coli. The purified C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins did not exhibit any differences in their Km(app) for the peptide substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) or Vmax(app), with respect to wild-type C alpha. All of the C subunit mutants displayed less than 2-fold changes in their Km(app) for ATP. The PKI alpha isoform displayed increased IC50 values for C alpha Y235S (71-fold), C alpha F239S (150-fold), and C alpha Y235S/F239S (1800-fold). Similarly, the PKI beta 1 protein showed increased IC50 values against the C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins, 9.4-, 11-, and 44-fold, respectively. In addition, the PKI alpha F10 codon was altered to an alanine codon, and this mutation decreased its ability to inhibit C alpha kinase activity, but did not affect its ability to inhibit C alpha Y235S/F239S. The mutation of Tyr235 and Phe239 to serines, however, did not alter the ability of the type II R subunit to inhibit phosphotransferase activity. These results suggest that C alpha Y235 and C alpha F239 are important for specific inhibition by both PKI alpha and PKI beta but not the type II R subunit and that mutations at these residues would be useful for in vivo analysis of C-PKI interactions.

Glutamic acid 203 of the cAMP-dependent protein kinase catalytic subunit participates in the inhibition by two isoforms of the protein kinase inhibitor.

Although the protein kinase inhibitors (PKIs) are known to be potent and specific inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase, little is known about their physiological roles. Glutamate 203 of the C alpha isoform (C alpha E203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKI alpha R15) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N., Taylor, S.S., and Sowadski, J. M. (1991) Science 253, 414-420). To investigate the role of C alpha E203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the C alpha E203 codon of the murine C alpha cDNA to alanine and glutamine codons. Initially, the C alpha E203 mutant proteins were expressed and purified from Escherichia coli. C alpha E203 is not essential for catalysis as all of the C subunit mutants were enzymatically active. The mutation of Glu203 did increase the apparent Km for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent Km for ATP. The Vmax(app) was not affected by the mutation of C alpha E203. The mutation of C alpha E203 compromised the ability of PKI alpha (5-24), PKI alpha, and PKI beta to inhibit phosphotransferase activity. PKI alpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with C alpha E203. The PKI alpha R15A mutant was reduced in its inhibition of C alpha. Preliminary studies of the expression of these C alpha mutants in COS cells gave similar results. These results suggest that the C alpha E203 mutants may be useful in assessing the role of PKI in vivo.

Alanyl-tRNA synthetase from Escherichia coli, Bombyx mori and Ratus ratus. Existence of common structural features.

Alanyl-tRNA synthetase from Escherichia coli, Bombyx mori and rat were examined with respect to the following functional and structural properties: the effect of substrates on sensitivity to proteolysis, secondary structure as determined by circular dichroism, amino acid composition and, in the case of the rat and insect enzymes, partial amino acid sequence determination on a 60-kDa C-terminal tryptic fragment. Digestion of the enzyme from all three sources with trypsin resulted in significant decline in aminoacyl-tRNA synthetase activity with little effect on pyrophosphate-exchange activity. In each case the presence of alanine and ATP together, but not separately, reduced the rate of digestion by trypsin; the largest effect was observed with the enzyme from rat liver. Trypsin digestion generated fragments of 47 kDa and 40 kDa with all three enzymes, but detection of significant quantities of the 47-kDa fragment from the rat enzyme required the presence of ATP and alanine. Trypsin digestion produced a fragment of 60 kDa with all three enzymes, but detection of significant quantities of this fragment with the bacterial enzyme required the presence of ATP and alanine. Limited sequence analysis of the 60-kDa fragment from the insect and rat enzymes indicated that trypsin cleaved both proteins at the same site to generate this species. Similar effects of substrates were observed when the enzymes were digested with chymotrypsin suggesting that the effects of substrates on protease sensitivity were not unique to trypsin. Circular dichroism spectra obtained for the three enzymes were qualitatively and quantitatively similar. There is some similarity in amino acid composition between the rat and insect enzymes.

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar.

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.

Identification of a developmentally regulated gene for a 140-kDa secretory protein in salivary glands of Chironomus tentans larvae.

Secretory proteins are synthesized in salivary glands of the insect, Chironomus tentans, and assemble in vivo into silk-like threads which aquatic larvae use to construct tubes for filter feeding and pupation. Thus far, all known secretory protein genes contain repetitious protein-coding sequences and are located in cytological structures known as Balbiani rings, giant puffs found on polytene secretory cell chromosomes. In this paper we describe the identification of another secretory protein gene which is comprised of repeated sequences; however, this gene is not located in a Balbiani ring. Two partial cDNA clones from a 3.6-kilobase pair poly(A)+ RNA were sequenced and found to contain two open reading frames for protein synthesis. Antibodies were raised against synthetic oligopeptides whose sequences were derived from these two open reading frames. An immunoaffinity-purified antibody for one of these peptides bound specifically to a 140-kDa secretory protein (sp140). The cDNA sequences contain tandem repeats of 42 base pairs which encode a repeat of 14 amino acids with a composition and oligopeptide sequence similar to other secretory proteins. The C. tentans genome contains about 70 copies of this 42-base pair repeat organized as a contiguous block of 3 kilobase pairs or less. The sp140 gene was mapped by in situ hybridization to polytene chromosome band I-17-B. Developmental studies of protein accumulation, steady-state levels of mRNA, and relative transcription rate suggested that the sp140 gene is developmentally regulated so that maximal expression is achieved during the prepupal stages of the fourth larval instar. Based upon these results we proposed that sp140 gene belongs to a prepupal class of secretory protein genes. While the sp140 gene shares structural and expression characteristics with other secretory protein genes, its unique chromosomal location shows that this multigene family is not restricted to Balbiani rings.

Control of the levels of alanyl-, glycyl-, and seryl-tRNA synthetases in the silkgland of Bombyx mori.

We have examined the levels of glycyl-, alanyl-, and seryl-tRNA synthetases and the levels of their corresponding translatable mRNAs in the posterior and middle silkglands of the silkworm, Bombyx mori. Analysis of Western blots reveals that the change in the abundance of these enzymes during the fifth instar in crude extracts derived from posterior and middle silkgland correlates with changes in enzymatic activity; most of the change in activity for seryl- and alanyl-tRNA synthetases can be accounted for by the corresponding change in enzyme concentration, while the apparent specific activity of glycyl-tRNA synthetase appears to be elevated in the posterior silkgland. Accompanying the changes in enzyme activity and enzyme concentration are changes in the levels of the corresponding mRNAs as determined by immunoprecipitation of in vitro translation products. The levels of all three enzymes are 10 to 20 times higher in the posterior and middle silkglands than in ovarian tissue. A form of alanyl-tRNA synthetase with a slightly higher apparent molecular weight is detected in the posterior silkgland early in the fifth instar and in ovarian tissue.

Glycyl- and alanyl-tRNA synthetases from Bombyx mori. Purification and properties.

Glycyl- and alanyl-tRNA synthetases have been purified from an extract of Bombyx mori posterior silk glands by a procedure that allows for the simultaneous isolation of both enzymes. Glycyl-tRNA synthetase is a dimer of Mr = 160,000 consisting of similar or identical subunits, whereas alanyl-tRNA synthetase is a monomer of Mr = 110,000 to 115,000. The abundance of these two enzymes in the posterior silk gland is consistent with the observed adaptation of this organ to the production of the silk protein, fibroin. The two enzymes are similar in oligomeric structure to the corresponding enzymes in Saccharomyces cerevisiae, but dissimilar from their counterparts in Escherichia coli.

In vivo and in vitro synthesis of the spore-specific proteins A and C of bacillus megaterium.

Pulse labeling of cells of Bacillus megaterium followed by cell disruption and immunoprecipitation has shown that the spore-specific Proteins A and C are synthetized only during a discrete time period in sporulation. At its maximum, the synthesis of the A- and C-proteins accounted for 5% of the protein being synthesized in vivo, but the mRNA for the A- and C-proteins had a lifetime no longer than that of other mRNAs translated at that time. No evidence was found for synthesis of Proteins A or C in high molecular weight precursor form, and essentially all of the newly synthesized A- and C-protein was found in the forespore. Isolation of total RNA from cells in various stages of growth and sporulation, translation of this RNA in a cell-free system from vegetative cells, and immunoprecipitation showed that the ability of cellular RNA to promote A- and C-protein synthesis in vitro was directly proportional to the rate at which the cells had been synthesizing Proteins A and C in vivo. These data indicate that synthesis of Proteins A and C during sporulation in B. megaterium is primarily under transcriptional control. The identity of the immunoprecipitated labeled material material synthesized in vitro with the A- and C-proteins was established by: 1) their co-migration on sodium dodecyl sulfate-polyacrylamide gels; 2) co-migration on high performance liquid chromatography of tryptic peptides from an [35S]methionine-labeled immunoprecipitate with the methionine-containing tryptic peptides of the A- and C-proteins; and 3) digestion of the labeled immunoprecipitate with a protease specific for the A- and C-proteins.

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.