scholarly journals Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase

1991 ◽  
Vol 278 (1) ◽  
pp. 203-209 ◽  
Author(s):  
A C Cannons ◽  
N Iida ◽  
L P Solomonson
Keyword(s):  
Cdna Clone ◽  
Spinacia Oleracea ◽  
Sequence Similarity ◽  
Cytochrome B5 ◽  
Binding Domain ◽  
Higher Plant ◽  
Expression Library ◽  
Reading Frame ◽  
Unicellular Green Alga ◽  
Partial Cdna Clone

A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed. A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells. pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells. The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame. The derived amino acid sequence of 318 amino acids has a 45-50% similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum). A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain. A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5. Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody. The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.

scholarly journals Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus.

1990 ◽  
Vol 111 (6) ◽  
pp. 2319-2326 ◽  
Author(s):  
M S Robinson
Keyword(s):  
Cdna Clone ◽  
Protein Complexes ◽  
Bovine Brain ◽  
Protein Sequencing ◽  
Cloning And Expression ◽  
Coated Vesicle ◽  
Expression Library ◽  
Coated Pits ◽  
Membrane Compartment ◽  
Partial Cdna Clone

Adaptins are the major components of adaptors, the protein complexes that link clathrin to transmembrane proteins (e.g., receptors) in coated pits and vesicles. The plasma membrane adaptor contains an alpha-adaptin subunit and a beta-adaptin subunit, while the Golgi adaptor contains a gamma-adaptin subunit and a beta'-adaptin subunit. A partial cDNA clone encoding gamma-adaptin was isolated from a bovine brain expression library by screening with antibodies, and was used to obtain a cDNA clone from a mouse brain library containing the full coding sequence. The identity of the clones was confirmed by protein sequencing. The deduced amino acid sequence of gamma-adaptin was found to be homologous to that of alpha-adaptin, with several stretches of identical amino acids or conservative substitutions in the first approximately 70 kD, and 25% identity overall. Weaker homology was seen between gamma- and beta-adaptins. Like both alpha- and beta-adaptins, gamma-adaptin has a proline and glycine-rich hinge region, dividing it into NH2- and COOH-terminal domains. A chimeric gamma-adaptin was constructed from the mouse and bovine cDNAs and transfected into Rat 1 fibroblasts. Immunofluorescence microscopy was carried out using an mAb which recognizes an epitope present on the chimera but not found on the rodent protein. The construct was found to have a distribution typical of endogenous gamma-adaptin. Using this transfection system, it should now be possible to exchange domains between alpha- and gamma-adaptins, to try to find out how adaptors are targeted to the appropriate membrane compartment of the cell, and how they recruit the appropriate receptors into the coated vesicle.

scholarly journals Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA

1987 ◽  
Vol 242 (2) ◽  
pp. 581-588 ◽  
Author(s):  
M R Jackson ◽  
L R McCarthy ◽  
D Harding ◽  
S Wilson ◽  
M W H Coughtrie ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Human Liver ◽  
Complete Sequence ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Base Pairs ◽  
Reading Frame ◽  
Partial Length ◽  
Liver Cdna Library ◽  
Udp Glucuronosyltransferase

A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets

Genome ◽  
1992 ◽  
Vol 35 (1) ◽  
pp. 133-139
Author(s):  
Charalambos Magoulas ◽  
Donal A. Hickey
Keyword(s):  
Drosophila Melanogaster ◽  
Cdna Clone ◽  
Sequence Similarity ◽  
Repetitive Sequences ◽  
Cdna Clones ◽  
Drosophila Genome ◽  
Significant Sequence Similarity ◽  
Reading Frame ◽  
Genomic Clones ◽  
Gene Libraries

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.Key words: Drosophila melanogaster, repetitive DNA, GCN repeats, polyalanine encoded domain.

scholarly journals Purification and initial characterization of the proteasome from the higher plant Spinacia oleracea.

1992 ◽  
Vol 267 (30) ◽  
pp. 21678-21684 ◽  
Author(s):  
M Ozaki ◽  
K Fujinami ◽  
K Tanaka ◽  
Y Amemiya ◽  
T Sato ◽  
...  
Keyword(s):  
Spinacia Oleracea ◽  
Higher Plant ◽  
Initial Characterization

Altered membrane lipase expression delays leaf senescence

2000 ◽  
Vol 28 (6) ◽  
pp. 775-777 ◽  
Author(s):  
J. Thompson ◽  
C. Taylor ◽  
T.-W. Wang
Keyword(s):  
Leaf Senescence ◽  
Cdna Clone ◽  
Transgenic Arabidopsis ◽  
Expression Library ◽  
Transgenic Arabidopsis Plants ◽  
Lipase Expression

A cDNA clone encoding a lipase that is upregulated in senescing leaves and flower petals has been isolated by screening an expression library. The abundance of the lipase mRNA increases as flowers and leaves begin to senesce, and expression of the gene is also induced by treatment with ethylene. Transgenic Arabidopsis plants in which levels of the senescence-induced lipase protein have been reduced show delayed leaf senescence.

Cloning and characterization of an electrogenic Na/HCO3− cotransporter from the squid giant fiber lobe

2007 ◽  
Vol 292 (6) ◽  
pp. C2032-C2045 ◽  
Author(s):  
Peter M. Piermarini ◽  
Inyeong Choi ◽  
Walter F. Boron
Keyword(s):  
Giant Axon ◽  
Predicted Amino Acid Sequence ◽  
Giant Fiber ◽  
Reading Frame ◽  
Excitable Membranes ◽  
Current Voltage ◽  
Partial Cdna ◽  
Partial Cdna Clone ◽  
Current Voltage Relationship

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na+-driven Cl-HCO3− exchanger, sqNDCBE—related to the SLC4 superfamily and cloned from giant fiber lobe cDNA—is the only HCO3−-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5′ and 3′ directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na+-coupled SLC4 transporters. However, when we measure pHi and membrane potential—or use two-electrode voltage clamping to measure currents—on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO3− cotransporter, NBCe. That is, exposure to extracellular CO2/HCO3− not only causes a fall in pHi, followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pHi recovery and current require HCO3− and Na+, and are blocked by DIDS. Furthermore, neither K+ nor Li+ can fully replace Na+ in supporting the pHi recovery. Extracellular Cl− is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.

An ubiquitously expressed gene 3.5 kilobases upstream of the glycerol-3-phosphate dehydrogenase gene in mice

1989 ◽  
Vol 9 (3) ◽  
pp. 935-945
Author(s):  
L A Johnston ◽  
M A Kotarski ◽  
D J Jerry ◽  
L P Kozak
Keyword(s):  
Sequence Similarity ◽  
Single Copy ◽  
Transcriptional Unit ◽  
Chromosome 15 ◽  
Glycerol Phosphate ◽  
Reading Frame ◽  
Dehydrogenase Gene ◽  
New Gene ◽  
Or Gene ◽  
Glycerol 3 Phosphate Dehydrogenase

While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.

scholarly journals Crystal structure of the N-terminal domain of VqsR fromPseudomonas aeruginosaat 2.1 Å resolution

2017 ◽  
Vol 73 (7) ◽  
pp. 431-436
Author(s):  
Qing He ◽  
Kang Wang ◽  
Tiantian Su ◽  
Feng Wang ◽  
Lichuan Gu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sequence Similarity ◽  
Transcriptional Regulator ◽  
Binding Domain ◽  
Structural Comparison ◽  
Homoserine Lactones ◽  
Terminal Domain ◽  
C Terminus ◽  
Common Motif ◽  
The Common

VqsR is a quorum-sensing (QS) transcriptional regulator which controls QS systems (las,rhlandpqs) by directly downregulating the expression ofqscRinPseudomonas aeruginosa. As a member of the LuxR family of proteins, VqsR shares the common motif of a helix–turn–helix (HTH)-type DNA-binding domain at the C-terminus, while the function of its N-terminal domain remains obscure. Here, the crystal structure of the N-terminal domain of VqsR (VqsR-N; residues 1–193) was determined at a resolution of 2.1 Å. The structure is folded into a regular α–β–α sandwich topology, which is similar to the ligand-binding domain (LBD) of the LuxR-type QS receptors. Although their sequence similarity is very low, structural comparison reveals that VqsR-N has a conserved enclosed cavity which could recognize acyl-homoserine lactones (AHLs) as in other LuxR-type AHL receptors. The structure suggests that VqsR could be a potential AHL receptor.

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