scholarly journals Cloning and Characterization of the Vitamin D Receptor from Xenopus laevis*

Endocrinology ◽  
1997 ◽  
Vol 138 (6) ◽  
pp. 2347-2353 ◽  
Author(s):  
Yan Chun Li ◽  
Clemens Bergwitz ◽  
Harald Jüppner ◽  
Marie B. Demay
Keyword(s):  
Vitamin D ◽  
Amino Acid ◽  
Xenopus Laevis ◽  
Vitamin D Receptor ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Amino Acid Level ◽  
Ion Homeostasis ◽  
Northern Analysis ◽  
Lower Vertebrate

Abstract The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species. The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor α, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expression is detected in many tissues including kidney, intestine, skin, and bone. During Xenopus development, xVDR messenger RNA first appears at developmental stage 13 (preneurulation), increasing to maximum at stages 57–61 (metamorphosis). Our data demonstrate that, in Xenopus, VDR expression is developmentally regulated and that the vitamin D endocrine system is highly conserved during evolution.

Salamander rods and cones contain distinct transducin alpha subunits

2000 ◽  
Vol 17 (6) ◽  
pp. 847-854 ◽  
Author(s):  
JAMES C. RYAN ◽  
SERGEY ZNOIKO ◽  
LIN XU ◽  
ROSALIE K. CROUCH ◽  
JIAN-XING MA
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Cellular Localization ◽  
Amino Acid Level ◽  
Single Copy ◽  
Lower Vertebrate ◽  
Rods And Cones ◽  
Sequence Identity ◽  
Outer Segments ◽  
Cone Pigments

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

The effects of D- and L-threo-chloramphenicol on the early development of the chick embryo

Development ◽  
1965 ◽  
Vol 13 (3) ◽  
pp. 341-356
Author(s):  
F. S. Billett ◽  
Rosalba Collini ◽  
Louie Hamilton
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Chick Embryo ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Animal Cells ◽  
Inhibit Protein Synthesis ◽  
Acid Complex ◽  
Amino Acid Complex ◽  
Bacterial Systems

In many bacterial systems chloramphenicol has been shown to inhibit protein synthesis (Hahn & Wisseman, 1951; Gale & Folkes, 1953). The precise mechanism of this inhibition is not clear, although the evidence suggests that the interaction of the soluble RNA-amino acid complex with the ribosomes is prevented because the attachment of the messenger RNA to the ribosomes is itself impaired (Lacks & Gros, 1959; Nathans & Lipman, 1961; Jardetsky & Julian, 1964; Julian & Jardetsky, 1964). In contrast to its effect on bacterial systems, chloramphenicol has been reported to have little or no action on the protein synthesis by cell-free extracts of mammalian cells (Rendi, 1959; Ehrenstein & Lipmann, 1961). A basis for this resistance has been proposed by Vazquez (1964), who finds that whereas bacterial ribosomes bind chloramphenicol, ribosomes from other organisms do not. Nevertheless, it cannot be stated with any confidence that chloramphenicol has no effect on the protein synthesis of animal cells.

scholarly journals Normalization of Mineral Ion Homeostasis by Dietary Means Prevents Hyperparathyroidism, Rickets, and Osteomalacia, But Not Alopecia in Vitamin D Receptor-Ablated Mice1

Endocrinology ◽  
1998 ◽  
Vol 139 (10) ◽  
pp. 4391-4396 ◽  
Author(s):  
Yan Chun Li ◽  
Michael Amling ◽  
Alison E. Pirro ◽  
Matthias Priemel ◽  
Jennifer Meuse ◽  
...  
Keyword(s):  
Vitamin D ◽  
Vitamin D Receptor ◽  
Ion Homeostasis

Cloning, sequencing and expression of stem cell factor (c-kit ligand) cDNA of brushtail possum (Trichosurus vulpecula)

1996 ◽  
Vol 8 (4) ◽  
pp. 789 ◽  
Author(s):  
PJ Greenwood ◽  
C Seamer ◽  
DJ Tisdall
Keyword(s):  
Stem Cell ◽  
Amino Acid ◽  
Stem Cell Factor ◽  
Biological Activities ◽  
Amino Acid Level ◽  
Amino Acid Sequences ◽  
Northern Analysis ◽  
Nucleotide Sequencing ◽  
Brushtail Possum ◽  
Factor C

By means of reverse transcription polymerase chain reaction (RT-PCR), three stem cell factor (SCF) cDNAs (822-738 bp in size) were amplified from brushtail possum ovarian poly (A)+ RNA. The largest and smallest of these cDNAs were cloned and sequenced. Characterization of these cDNAs has revealed that possum SCF has approximately 75% and 66% homology to SCF of eutherian mammals at the nucleotide level and the predicted amino acid level respectively. Nucleotide sequencing shows that the 738-bp cDNA represents an mRNA splice variant, equivalent to that found in eutherian mammals, in which an exon (84 bp) encoding a potential proteolytic cleavage site is removed. Comparison of the predicted possum SCF amino acid sequence with the predicted SCF amino acid sequences from eutherian mammals reveals conservation of all cysteine residues and 3 of 4 potential N-linked glycosylation sites. In addition, the hydropathicity profile of the possum SCF protein is similar to that of eutherian SCF suggesting that protein conformation is conserved. Northern analysis was used to characterize possum SCF gene expression in adult ovary and testis. A major transcript of 9 kb was observed in both ovarian and testicular tissue. The conservation of the SCF gene and its predicted protein, suggests that SCF in the possum has similar biological activities to SCF in eutherian mammals.

A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division

1994 ◽  
Vol 107 (1) ◽  
pp. 253-265 ◽  
Author(s):  
I.T. Todorov ◽  
R. Pepperkok ◽  
R.N. Philipova ◽  
S.E. Kearsey ◽  
W. Ansorge ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Amino Acid ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Nuclear Protein ◽  
Amino Acid Level ◽  
S Phase ◽  
Significant Similarity

Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2. Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.

scholarly journals Cloning and expression of a cDNA encoding a mouse brain orphanin FQ/nociceptin precursor

1996 ◽  
Vol 315 (1) ◽  
pp. 11-13 ◽  
Author(s):  
Ying-Xian PAN ◽  
Jin XU ◽  
Gavril W. PASTERNAK
Keyword(s):  
Amino Acid ◽  
Mouse Brain ◽  
Single Gene ◽  
Amino Acid Level ◽  
Cloning And Expression ◽  
Northern Analysis ◽  
Orphanin Fq ◽  
Amino Acid Peptide ◽  
Reverse Transcription Pcr ◽  
Mouse Sequence

By using a reverse transcription–PCR approach we have cloned a peptide precursor from mouse brain which contains the sequence of orphanin FQ/nociceptin. The mouse sequence of orphanin FQ/nociceptin is identical at the amino acid level with that isolated from rat and porcine brain. Northern analysis of the mRNA encoding the precursor reveals a single band of approx. 1 kb, with the highest levels in the brain and much lower levels in kidney and spleen. Southern analysis is consistent with a single gene. The precursor peptide from mouse contains two other putative peptides. Upstream from the orphanin FQ/nociceptin is a 41-amino-acid peptide which is almost identical, except for a six-amino-acid insertion, with the corresponding 35-amino-acid peptide predicted from the rat sequence. Interestingly, the mouse contains a triple AEPGAD repeat within this peptide that is not seen in the rat sequence. Downstream from the orphanin FQ/nociceptin sequence is another 17-amino-acid peptide which is identical with that found in the rat.

scholarly journals Molecular characterisation and hormone-dependent expression of the porcine whey acidic protein gene

1998 ◽  
Vol 20 (1) ◽  
pp. 27-35 ◽  
Author(s):  
KJ Simpson ◽  
P Bird ◽  
D Shaw ◽  
K Nicholas
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Level ◽  
Whey Acidic Protein ◽  
Acidic Protein ◽  
Northern Analysis ◽  
Major Protein ◽  
Sds Page

A 17.5 kDa protein was isolated from porcine whey by reverse phase HPLC and identified as a putative whey acidic protein (WAP) homologue by sequencing 35 and 40 amino acid residues of the amino- and carboxy-terminus respectively. Degenerate oligonucleotides to both of these amino acid sequences were designed and used in reverse transcriptase PCR with RNA from lactating porcine mammary gland as a template. A 162 bp PCR fragment was detected and sequenced. Compilation of the deduced and determined amino acid sequence revealed a protein of 111 amino acids, which had approximately 75, 50, 40 and 35% similarity at amino acid level to camel, rabbit, rat and mouse WAP respectively. It also included the four-disulphide core characteristic of all WAP proteins and most Kunitz-type protease inhibitors. This provides the first unequivocal evidence for WAP secretion in the pig. SDS PAGE analysis of the whey fraction showed that WAP is secreted as a major protein in sow's milk from farrowing to weaning. The molecular mass of WAP in SDS PAGE was significantly greater than the 11.7 kDa determined from amino acid sequence, indicating that porcine WAP is possibly glycosylated. Northern analysis detected a single mRNA transcript of approximately 600 bp in porcine RNA from the mammary gland of lactating sows. To examine the hormone-regulated expression of the WAP gene the mammary glands of sows at day 90 of pregnancy were biopsied and explants cultured for 3 days in the presence of various combinations of porcine insulin (I), cortisol (F) and porcine prolactin (P). Northern analysis of RNA extracted from the tissue indicated that WAP gene expression was barely detectable in the mammary gland prior to culture and there was no increment in explants cultured in the presence of I and F. However, a significant increase in the accumulation of WAP mRNA was observed in explants cultured in I, F and P. A similar result was observed for beta-casein and alpha-lactalbumin gene expression.

HUMAN NEUTROPHILS EXPRESS MESSENGER RNA OF VITAMIN D RECEPTOR AND RESPOND TO 1α,25-DIHYDROXYVITAMIN D3

2002 ◽  
Vol 24 (3) ◽  
pp. 335-347 ◽  
Author(s):  
Katsushi Takahashi ◽  
Yasunori Nakayama ◽  
Hideki Horiuchi ◽  
Tomohiro Ohta ◽  
Keiji Komoriya ◽  
...  
Keyword(s):  
Vitamin D ◽  
Vitamin D Receptor ◽  
Messenger Rna ◽  
Human Neutrophils ◽  
Dihydroxyvitamin D3

scholarly journals Targeted Deletion of Vitamin D receptor Gene in Mammalian Cells by CRISPR/Cas9 Systems

2016 ◽  
Author(s):  
Tao zhang ◽  
Ling Wang ◽  
Kun Xu ◽  
Chonghua Ren ◽  
Zhongtian Liu ◽  
...  
Keyword(s):  
Vitamin D ◽  
Vitamin D Receptor ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Chromosome Region ◽  
Receptor Gene ◽  
Vdr Gene ◽  
Targeted Deletion ◽  
Guide Rna ◽  
Conserved Regions

CRISPR/Cas9 system has become a new versatile technology for genome engineering. It utilizes a single guide RNA (sgRNA) to recognize target sequences in genome function, and activates Cas9 endonucleases to cut the locus. In this study, we designed two target sites from conserved regions of vitamin D receptor (VDR) gene in mammalian cells, which cover more than 17 kb of chromosome region depending on the species. The efficacy of single sgRNA mediated gene specific modification was about 22% to 36%. Concurrently, targeted deletions of the intervening genomic segments were generated in chromosomes when the two sgRNAs worked simultaneously. The large genomic DNA segments ranging from 17.8Kb to 23.4 Kb could be precisely deleted in human and mouse chromosomes. Furthermore, the expression level of 24-hydroxylase (CYP24A1) regulated by VDR was significantly increased in cells treated with VDR CRISPR/Cas9 vectors. This study showed that CRISPR/Cas9 system can be employed to generate large genomic segment deletions in different species, providing sgRNAs are designed within conserved regions.

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