The putative gymnosperm plant defensin polypeptide (SPI1) accumulates after seed germination,is not readily released,and the SPI1 levels are reduced in Pythium dimorphum-infected spruce roots

The putative plant defensin SPI1 cDNA from the conifer Norway spruce (Picea abies) is the only known plant defensin-like sequence from a gymnosperm. The predicted translational product SPI1 was not detected in the embryo or other parts of the seed by means of antibodies, but it accumulated in the root cortex after germination. In roots of seedlings infected with the root pathogenic oomycete Pythium dimorphum and the blue stain fungus Ceratocystis polonica, variable levels of SPI1 was detected during the first day as a response to the infection, however a significant increase was seen as an initial response to the root-rot fungus Heterobasidion annosum. After the first day of infection, the amount of SPI1 polypeptide was dramatically reduced in response to either of the pathogens, but not in response to the ectomycorrhizal fungus Laccaria bicolor. During the same time of infection, extensive damage to cortical root cells resulted from the infecting pathogens, but not from the mycorrhiza. These results indicate that pathogens may reduce the level of SPI1 by suppressing its expression, but may also reduce the SPI1 level by invading and disrupting the root cortical cells or by a combination of these mechanisms.     

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

Summary A cDNA encoding -ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned fromCuphea lanceolata. This cDNA of 1276 by codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp -ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the -ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, -ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced inEscherichia coli, was isolated and proved to possess -ketoacyl-ACP reductase activity. Hybridization studies revealed that inC. lanceolata -ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.     

Isolation of repetitive clones from human muscle cDNA library

Human fetal muscle cDNA library was screened with a-myosin heavy chain gene fragment containing Alu sequences. Two cDNA clones AI and BII with 1.8 and 3 kb inserts respectively were chosen for further characterization by means of RNA and DNA hybridization procedures and sequencing. The clones appeared to contain repetitive sequences as well as single copy regions. They are actively transcribed in different stages of myogenic development but not in the liver. DNA sequence analysis of short stretches from both clones revealed no sequence homology to any other published DNA sequences.     

Isolation and characterization of lipid globules from the zoospores of Blastocladiella emersonii

Lipid globules were isolated and characterized both chemically and morphologically. They were composed mainly of triglyceride and free sterol, which accounted for over 90% of the total globule content. Smaller amounts of diglyceride, carotenoid, free fatty acid, phospholipid and protein were found. No sterol esters or monoglycerides were detected. Morphologically, the isolated lipid globules resembled the lipid globules in situ. They were spherical, 0.4–1.5 m in diameter and lacked a trilaminar membrane.Non-Standard Abbreviations PL phospholipids - TG triglycerides - FS Tree sterols - DG diglycerides - SE sterol esters - MG monoglycerides     

The structure of aPhaseolus vulgaris cDNA encoding the iron storage protein ferritin

A cDNA containing the entire coding region for the iron storage protein ferritin has been isolated from the French bean plant,Phaseolus vulgaris L. cv. Tendergreen. Ferritin protein was purified from young leaves and shoot meristem tissue and used to raise antisera in mice. A gt11 cDNA library was constructed from seed-derived poly(A)⁺ RNA, and screened with the mouse anti-ferritin serum. A 1.2 kb immunopositive phage DNA insert was isolated and sequenced. The derived amino acid sequence shows substantial similarity with other ferritin sequences. The 5 untranslated region contains two out-of-frame AUG codons, a region of extreme pyrimidine composition bias and potentially stable secondary structure.     

The feeding ecology of a carnivorous plant (Pinguicula nevadense): prey analysis and capture constraints

Summary The taxonomic composition and size of arthropods captured by Pinguicula nevadense, an endemic carnivorous plant of the high-mountain zone of the Sierra Nevada (southern Spain), are analysed. The actual prey of P. nevadense and the available arthropods trapped by mimic-traps are compared, in order to identify the capture constraints of the plant. The results show that P. nevadense captures various arthropod taxa. Winged insects, especially Nematocera, make up the main component of the diet. The range of prey sizes in all P. nevadense populations studied is similar. The taxonomic composition of arthropods trapped by the mimic-traps is similar to that of the actual prey of P. nevadense. However, the plant captures prey only below a specific size threshold. These size constraints appear to be the principal factor determining the actual prey of this carnivorous plant.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after -ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5 and 3 ends of dTnp1 together with a perfect palindrome located after the 5 inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Structure and expression of a cDNA encoding a histone H2A from Euglena gracilis

Screening of a gt11 cDNA expression library of Euglena gracilis with antibodies directed against histones H2 from maize resulted in the isolation of a full-length cDNA for a histone H2A. The open-reading frame of 408 bp corresponded to a protein of 136 amino acid residues (14 kDa). Despite the presence of a poly(A) tail, which is typical of plant histone mRNA but not of animal histone mRNA, the size of the deduced protein and its percentage of homology were closer to animal histone H2As than to plant or lower eukaryotic histone H2A.Sequence alignment revealed that the Euglena H2A protein was characterized by a shorter C-terminus and a N-terminus which extended 10 residues past the animal H2A.     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

Isolation of a raw starch-binding fragment from barley alpha-amylase

Barley -amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with -cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel -sheets in domain C and the ₇-and ₈-helices of the (/)₈ domain.     

Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H.Calcium and Cell Function III: 161–188, 1982). This active preparation consists of three related species containing residues 1–290, 1–296, and 1–298 of the 44.7 kD -subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740–11745, 1991). Good recoveries of catalytic activity — with varying degrees of calcium dependence — result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein.Physical characterization shows that the 33 kD chymotryptic fragment is globular, withs _20,w=2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylasea andb, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R.Biochem. Biophys. Res. Comm. 174: 344–350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[-P³²] ATP results in the reaction of 0.7 mol³²P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of³²P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser³⁰ while in the latter an additional reaction takes place at Ser⁸¹. The phosphopeptides correspond to sequences occurring in the -subunit of phosphorylase kinase.Abbreviations cAMP adenosine 3(,5(-cyclic monophosphate - NaDodSO₄ sodium dodecyl sulfate - Mops 3-(N-morpholino)propanesulfonic acid - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N(,N(-tetraacetic acid - ANS 1-anilinonapththalene-8-sulfonate - TPCK N-tosyl phenylalanyl chloromethyl ketone - TLCK N-tosyllysyl chloromethyl ketone - PTH phenylthiohydantoin The expected two to three fold increases in fluorescence intensity occur when assays are conducted with solutions containing 20 mM Tris chloride and no glycerophosphate.     

Role of protein phosphorylation in regulation of brainL-glutamate decarboxylase activity

In the brain, the -aminobutyric acid (GABA) level is primarily controlled by the activity of its synthesizing enzyme,L-glutamate decarboxylase (GAD). At present, mechanisms responsible for regulation of GAD activity remain largely unknown. Here we report that GAD activity is inhibited by conditions favoring protein phosphorylation, and this inhibition can be reversed by phosphatase treatment. Furthermore, this inhibition appears to result from the suppression of a Ca²⁺-dependent phosphatase. Phosphorylation of GAD is demonstrated by direct incorporation of³²P into the GAD protein. These results suggest that GAD activity in the brain is inhibited by phosphorylation and activated by dephosphorylation. A model for regulation of GABA synthesis related to neuronal excitation is discussed.     

Characterization of a predominantly pistil-expressed gene encoding a γ-thionin-like protein of Petunia inflata

We isolated a cDNA clone from a pistil cDNA library of Petunia inflata which encodes a protein, PPT, with sequence similarity to -thionins. Characterization of a genomic clone containing a PPT gene revealed the presence of a single intron. Northern analysis revealed that the PPT gene was predominantly expressed in the pistil during all stages of flower development. Since thionins have been implicated in plant defense against pathogens, PPT may play a role similar to that of other defense-related proteins found in the pistil, defending the pistil against pathogen infection.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

The protein sequence homology of gamma-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of -crystallin were found to be N-terminally blocked. -Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified -crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between -crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp -II, rat 2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat -crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one // superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.