PHH1, a novel gene from Arabidopsis thaliana that encodes a protein similar to plant blue-light photoreceptors and microbial photolyases

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity (54% protein identity), including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5′-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1ΔC-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein.     

Isolation and characterization of homologues of plant blue-light photoreceptor (cryptochrome) genes from the fern Adiantum capillus-veneris

Many blue-light mediated physiological responses have been studied in the fern Adiantum capillus-veneris. We have isolated genomic clones encoding sequences similar to those encoding blue-light photoreceptors (cryptochromes) in higher plants using the Arabidopsis CRY1 cDNA as a probe, and these positive clones fall into five independent groups. Using RACE procedures, we obtained full-length cDNA sequences for three of these five groups. The deduced amino acid sequences include the photolyase-homologous domain in the N-terminal half, and they also contain a C-terminal extension of about 200 amino acids in length. These structural features indicate that the genes indeed encode Adiantum cryptochromes and represent a small gene family having at least three members. Received: 16 February 1998 / Accepted: 26 April 1998     

Production of synthetic spider dragline silk protein in Pichia pastoris

The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation. Genes up to 3000 codons in length or longer could be expressed with no evidence of the prevalent truncated synthesis observed for similar genes in Escherichia coli, though genes longer than 1600 codons were expressed less efficiently than shorter genes. Silk-producing P. pastoris strains were stable without selection for at least 100 doublings. Received: 4 March 1996 / Received revision: 26 June 1996 / Accepted: 12 August 1996     

Characterization of photolyase/blue-light receptor homologs in mouse and human cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).     

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1 ⁺ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron. Received: 26 April 1996 / Accepted: 22 August 1996     

Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp. KOD1 possesses a mosaic structure showing features of various oxidoreductases

Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB, encoding α and β subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationaly coupled. Sequence analysis showed the C-terminal region of the α subunit to have a typical ferredoxin-type [4Fe-4S] cluster motif (CXXCXXCXXCXXXCP), which is similar to that present in the δ subunits of other oxidoreductases such as pyruvate ferredoxin oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the α subunit of KOD1-IOR has a mosaic structure composed of features characteristic of the α, β and δ subunits from POR and VOR. KOD1-IOR was overproduced in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant IOR was 70° C and the half-life of this enzyme in the presence of air was 15 min at 25° C. Received: 25 September 1996 / Accepted: 20 December 1996     

Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Cloning, sequencing and prokaryotic expression of cDNAs for antifreeze protein family from beetle Tenebrio molitor

Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR. Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank. The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E. coli BL21 to induce a GST fusion protein by IPTG. SDSPAGE analysis for the fusion protein shows a band of 38 kDa. pCDNA3-tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA. The titer of the antibody was 1:2000.Western blotting analysis shows that the antiserum was specifically against the antifreeze protein. Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1532-1540 [译自: 遗传]     

Rice has two distinct classes of protein kinase genes related to SNF1 of Saccharomyces cerevisiae , which are differently regulated in early seed development

We have isolated five cDNA clones (osk1–5) for protein kinases from rice which are related to SNF1 protein kinase of Saccharomyces cerevisiae. Based on the sequence homology, these cDNAs can be classified into two groups, group 1 (osk1) and group 2 (osk2–5). The products of these genes were demonstrated to be functional SNF1-related protein kinases by in vitro and in vivo experiments. Recombinant proteins expressed from both groups of genes were fully active as protein kinases and could phosphorylate SAMS peptide, a substrate specific for the SNF1/AMPK family, as well as themselves (autophosphorylation). Moreover, expression of osk3 cDNA in yeast snf1 mutants restored SNF1 function. Northern blot analyses showed differential expression of these two gene groups; group 1 is expressed uniformly in growing tissues (young roots, young shoots, flowers, and immature seeds), whereas group 2 is strongly expressed in immature seeds. SNF1-related protein kinases have been reported from different plant species, such as rye, barley, Arabidopsis, tobacco, and potato, while the type of gene strongly expressed in immature seeds is known only in cereals such as rye, barley, and, from our findings, in rice. Expression levels of the group 2 genes were further analyzed in seeds during seed maturation. Expression is transiently increased in the early stages of seed maturation and then decreases. The expression peak precedes those of the sbe1 and waxy genes, which are involved in starch synthesis in rice. Taken together, these findings suggest that group 2 OSK genes play important roles in the early stages of endosperm development in rice seeds. Received: 30 April 1998 / Accepted: 20 August 1998     

At UBP3 and At UBP4 are two closely related Arabidopsis thaliana ubiquitin-specific proteases present in the nucleus

The ubiquitin-specific proteases (UBPs) are a class of enzymes vital to the ubiquitin pathway. These enzymes cleave ubiquitin at its C-terminus from two types of substrates containing (i) ubiquitin in an α-amino linkage, as found in the primary ubiquitin translation products, polyubiquitin and ubiquitin-ribosomal fusion proteins, or (ii) ubiquitin in an ɛ-amino linkage, as found in multiubiquitin chains either unattached or conjugated to cellular proteins. We have isolated cDNAs for two Arabidopsis thaliana genes, AtUBP3 and AtUBP4, which encode UBPs that are 93% identical. These two cDNAs represent the only two members of this subgroup and encode the smallest UBPs described to date in any organism. Using in vivo assays in Escherichia coli that allow the coexpression of a UBP with a putative substrate, we have shown that AtUBP3 and AtUBP4 can specifically deubiquitinate the artificial substrate Ub-X-β-gal but cannot act upon the natural α-amino-linked ubiquitin fusions Arabidopsis Ub-CEP52 and Arabidopsis polyubiquitin. Affinity-purified antibody prepared against AtUBP3 expressed in E. coli recognizes both AtUBP3 and AtUBP4. AtUBP3 and/or AtUBP4 are present in all Arabidopsis organs examined and at multiple developmental stages. Subcellular localization studies show that AtUBP3 and/or AtUBP4 are present in nuclear extracts. Possible physiological roles for these UBPs are discussed. Received: 14 November 1996 / Accepted: 6 February 1997     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway. Received: 15 August 1996 / Accepted: 23 October 1996     

Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes

Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases. Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds to theArabidopsis thaliana gene,cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes. The cDNA shares 44% amino acid identity with theEscherichia coli cytidine deaminase but only 26 and 27% identity with human and yeast enzymes. A unique zinc-binding domain of the Ecoli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that of the Ecoli enzyme. In this model, the active site zinc is coordinated by His⁷³, Cys¹⁰³, Cys¹⁰⁷, and an active site hydroxyl. Additional residues that participate in catalysis, Asn⁶⁴, Glu⁶⁶, Ala⁷⁸, Glu⁷⁹, and Pro¹⁰², are conserved between the Arabidopsis and Ecoli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the Ecoli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to theA thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His₆ tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (K_m = 226 μand 2’-deoxycytidine (K_m= 49 μM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP. journal Paper Number J-18324 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 3340.     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. Received: 14 January 1998 / Accepted: 31 March 1998     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.