Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae

By the complementation of a yeast mutant defective in myo-inositol transport (Nikawa, J., Nagumo, T., and Yamashita, S. (1982) J. Bacteriol. 150, 441-446), we isolated two myo-inositol transporter genes, ITR1 and ITR2, from a yeast gene library. The ITR1 and ITR2 genes contained long open reading frames capable of encoding 584 and 612 amino acids with calculated relative molecular masses of 63,605 and 67,041, respectively. The sequence similarity between the ITR1 and ITR2 products was extremely high, suggesting that the two genes arose from a common ancestor. Both gene products show significant sequence homology with a superfamily of sugar transporters, including human HepG2 hepatoma/erythrocyte glucose transporter and Escherichia coli xylose transporter. Hydropathy analysis indicated that the ITR1 and ITR2 products are both hydrophobic and contain 12 putative membrane-spanning regions. Thus, yeast myo-inositol transporters could be classified into the sugar transporter superfamily. Gene disruption and tetrad analysis showed that yeast cells contain two separate myoinositol transporters. The ITR1 product was the major transporter and the ITR2 product the minor one in cells grown in minimum medium containing glucose. Northern blot analysis showed that ITR1 mRNA was much more abundant than ITR2 mRNA. The previously isolated myo-inositol transport mutant was determined to be defective in ITR1.     

Isolation and characterization of two forms of malate dehydrogenase from human placenta

• 1.1. The purification and characterization of the cytoplasmic and mitochondrial forms of malate dehydrogenase from human placenta are described.
• 2.2. Both enzymes are composed of two subunits and have similar molecular weights and similar pH optima.
• 3.3. However, they differ with respect to thermal stability, excess substrate inhibition and electrophoretic mobility.

     

Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus

Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the K_m values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane.     

Isolation and characterization of human thioredoxin-encoding genes

Thioredoxin (Trx) has recently been demonstrated to be an essential component of the early pregnancy factor activity of pregnancy serum. Here, we report the structure and sequence of human Trx-encoding genes (Trx) by analysis of genomic clones. The Trx gene extends over 13 kb and consists of five exons encoding a 12-kDa protein. A 700-bp fragment upstream from the start codon functions as a promoter when inserted in front of a human growth hormone-encoding reporter gene in tissue-culture cells. This promoter region is very G + C rich and does not contain a classical TATA or CCAAT box, but has three consensus sequences for high-affinity Sp1 binding. Southern analysis demonstrated the presence of several Trx genes in the human genome. The number includes at least one inactive copy as shown by the isolation and sequencing of an inactive pseudogene.     

Isolation and characterization of two human H1 histone genes within clusters of core histone genes

Two human H1 histone genes, termed H1.3 and H1.4, were isolated from two cosmid clones. The H1.4 gene is associated with an H2B gene, whereas genes coding for all four core histones are located in the vicinity of the H1.3 gene. This cluster arrangement was found both in the two cosmid clones and on overlapping bacteriophage clones isolated from an EMBL3 library. In continuation of our previous analysis of two human H1 genes, this analysis raises the number of completely sequenced H1 histone genes within clusters of core histone genes to four.     

Isolation and characterization of two wound-regulated tomato extensin genes

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library byin situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro₄-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro₄-Tyr₃-Lys-Ser-Pro₄-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro₄ repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro₄-Ser-Pro-Ser-Pro₄-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.     

Isolation and characterization of two distinct forms of protein kinase C

Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.     

Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.     

Isolation and characterization of two distinct mannan-binding proteins from rat serum

Two binding proteins, which are specific for mannose and N-acetylglucosamine, were isolated from rat serum to homogeneity. The minor component [serum mannan-binding protein I (S-MBP-I)] was indistinguishable from rat liver mannan-binding protein (L-MBP). S-MBP-I had a molecular mass of about 200 kDa and consisted of about six identical 32-kDa subunits; the molecule had a collagen-like structure, and its properties were identical to those of L-MBP. S-MBP-I was also indistinguishable from L-MBP in immunochemical reactivity. Furthermore, the sequence of 15 NH2-terminal amino acids of S-MBP-I was identical to that of L-MBP, the complete primary structure of which has been elucidated [Drickamer, K., Dordal, M. S., and Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., and Yamashina, I. (1987) J. Biochem. 101, 135-144]. The major component (S-MBP-II) had a molecular mass of about 650 kDa and consisted of about 20 identical 31-kDa subunits; it was immunochemically distinct from L-MBP and S-MBP-I, although the molecule had a collagen-like structure similar to L-MBP and S-MBP-I. Metabolic studies using [3H]leucine showed that S-MBP-II is a typical plasma protein turning over with a half-life of 1.6 days. S-MBP-I was unusual in its late appearance and rapid turnover rate in plasma. These results, together with the fact that L-MBP decayed with biphasic curves, suggest that a part of L-MBP is leaked from liver into plasma in the form of S-MBP-I.     

Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast

Summary Yeast phosphofructokinase is an octamer composed of two different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology.     

Two genes encode distinct glutamate decarboxylases

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.     

Isolation of two distinct type I angiotensin II receptor genes.

A rat genomic Southern blot, probed with a type I angiotensin II receptor probe, demonstrated that two highly homologous type I angiotensin II receptors were present. A rat genomic library was subsequently screened and four clones were isolated. From restriction mapping, differential hybridization, polymerase chain reaction amplification and sequence analyses we have determined that there are two unique type I angiotensin II receptor genes. The first of these genes corresponds to the published rat vascular complementary DNA sequence; the second, corresponds to a novel receptor not previously described.     

Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase

Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD(+) binding site within human GDH encoded by the synthetic human GDH gene and expressed in Escherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+) binding site. Amino acid sequencing and compositional analysis identified Glu(279) as the site of photoinsertion into human GDH, suggesting that Glu(279) is located at or near the NAD(+) binding site. The importance of the Glu(279) residue in the binding of NAD(+) was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu(279) has no effects on the expression or stability of the different mutants. The K(m) values for NAD(+) were 10-14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V(max) values were similar for wild-type and mutant GDHs. The efficiency (k(cat)/K(m)) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K(m) values for NAD(+). In contrast to the K(m) values for NAD(+), wild-type and mutant GDHs show similar K(m) values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu(279) is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu(279) plays an important role for efficient binding of NAD(+) to human GDH.     

Isolation and characterization of two knotted-like homeobox genes from tomato

Homeobox genes are known to play a role in developmental regulation. The knotted-like homeobox (knox) genes fall into two classes. The class I knox genes like kn1, stm1, and knat1 are involved in maintaining meristem identity in cells. The function of class II knox genes is at yet undetermined. We have characterized two knox genes from tomato. LeT6 and LeT12 map to distinct chromosome locations that are different from the location for a recently cloned knox gene from tomato, tkn1, confirming that plant homeobox genes are not clustered on chromosomes. These genes have a distinct expression pattern. Unlike other class I kn1-like genes, LeT6 is expressed in developing lateral organs and developing ovaries in flowers. LeT12 is more ubiquitously expressed in the mature plant. RNA in situ localization data suggest that both these genes may have a role to play in formative events in ovule and embryo morphogenesis.     

Isolation and characterization of two cDNAs from atlantic cod encoding two distinct psychrophilic elastases

The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50–64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.     

Isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescence

DNA topoisomerases which remove superhelical turns in closed circular DNA have been isolated from cauliflower inflorescences using polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex or CM-cellulose and DNA-cellulose. Two distinct enzymes, topoisomerase-I and ATP-dependent topoisomerase, were separated clearly by CM-Sephadex or CM-cellulose, and partially characterized using agarose gel electrophoresis with plasmid pBR322 DNA. Topoisomerase-I acts like other eucaryotic DNA topoisomerases in the absence of ATP, is stimulated by spermidine and inhibited by EDTA. The ATP-dependent topoisomerase acts like topoisomerase-I only in the presence of ATP in the reaction medium, is inhibited by spermidine and EDTA, and does not introduce supertwists into closed duplex DNA or produce catenate aggregates under the present reaction conditions.