Identification and expression of a regeneration-specific homeobox gene in the newt limb blastema

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 489-496 ◽  
Author(s):  
R. Brown ◽  
J.P. Brockes
Keyword(s):  
Retinoic Acid ◽  
Amino Acid ◽  
Pattern Formation ◽  
Amino Acid Sequence ◽  
Homeobox Gene ◽  
Growth Zone ◽  
Epithelial Tissue ◽  
Cdna Clones ◽  
Time Points ◽  
Newt Limb

Adult urodele amphibians are able to regenerate their limbs through the formation of a blastema, a growth zone of undifferentiated mesenchymal cells that arises locally at the plane of amputation. In an effort to define genes involved in pattern formation by the blastema, we isolated from a newt forelimb blastema library cDNA clones that identify a homeobox gene termed NvHbox 2. The amino acid sequence of the homeodomain is identical to that of the recently identified human HOX-4f gene (Acampora et al. 1989) and of the mouse Hox-5.5 (Dolle et al. 1989). NvHbox 2 is expressed in the limb blastema as a transcript of 3.4 kb that is not detectable in the normal limb. Analysis by RNAase protection demonstrates expression in limb and tail blastemas, but not in any of the adult tissues or organs tested. In the limb blastema NvHbox 2 was expressed in mesenchymal but not epithelial tissue. When matched and normalised samples of RNA from proximal (mid-humerus) and distal forelimb (mid-radius) blastemas were compared, the level of expression of NvHbox 2 was found to be 3- to 5-fold higher proximally. At two time points after injection of a proximalising dose of retinoic acid, the level of expression in a distal blastema was not increased in comparison to controls.

Analysis of Hox-4.5 and Hox-3.6 expression during newt limb regeneration: differential regulation of paralogous Hox genes suggest different roles for members of different Hox clusters

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1397-1407 ◽  
Author(s):  
H.G. Simon ◽  
C.J. Tabin
Keyword(s):  
Hox Genes ◽  
Limb Regeneration ◽  
Differential Regulation ◽  
Epithelial Tissue ◽  
Cdna Clones ◽  
Rnase Protection ◽  
Adult Kidney ◽  
Early Expression ◽  
Hox Clusters ◽  
Newt Limb

Adult urodele amphibians can regenerate their limbs and tail. Based on their roles in other developing systems, Hox genes are strong candidates for genes that play a role in regulating pattern formation during regeneration. There are four homologous clusters of Hox genes in vertebrate genomes. We isolated cDNA clones of two newt homeobox genes from homologous positions within two Hox clusters; Hox-4.5 and Hox-3.6. We used RNase protection on nonamputated (normal) and regenerating newt appendages and tissue to compare their transcriptional patterns. Both genes show increased expression upon amputation with similar kinetics. Hox-4.5 and Hox-3.6 transcription is limited to the mesenchymal cells in the regenerates and is not found in the epithelial tissue. In addition to regenerating appendages, both genes are transcriptionally active in adult kidney of the newt. Striking differences were found in the regulation of Hox-4.5 and Hox-3.6 when they were compared in unamputated limbs and in regenerating forelimbs versus regenerating hindlimbs. Hox-4.5 is expressed in the blastema of regenerating fore- and hindlimbs, but Hox-4.5 transcripts are not detectable in normal limbs. In contrast, Hox-3.6 transcripts are found exclusively in posterior appendages, but are present in normal as well as regenerating hindlimbs and tails. Hox-4.5 is also expressed at a higher level in proximal (mid-humerus) regenerates than in distal ones (mid-radius). When we proximalized the positional memory of a distal blastema with retinoic acid, we find that the early expression level of Hox-4.5 is also proximalized. When the expression of these genes is compared to the expression of two previously reported newt Hox genes, a consistent pattern emerges, which can be interpreted in terms of differential roles for the different Hox clusters in determining regenerative limb morphology.

The NH2 -terminal amino acid sequence of cellular retinoic-acid binding protein from rat testis

FEBS Letters ◽  
1981 ◽  
Vol 135 (1) ◽  
pp. 70-72 ◽  
Author(s):  
Ulf Eriksson ◽  
Johan Sundelin ◽  
Lars Rask ◽  
Per A. Peterson
Keyword(s):  
Retinoic Acid ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Protein ◽  
Terminal Amino Acid ◽  
Rat Testis ◽  
Acid Binding Protein ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

scholarly journals Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase

1995 ◽  
Vol 305 (2) ◽  
pp. 439-444 ◽  
Author(s):  
T M Johnson ◽  
H P Kocher ◽  
R C Anderson ◽  
G M Nemecek
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heterologous Expression ◽  
Stop Codon ◽  
Expression System ◽  
Breast Muscle ◽  
Cdna Clones ◽  
Carnitine Acetyltransferase ◽  
Western Blots ◽  
Pigeon Liver

Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5′ and 3′ ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3′-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.

scholarly journals Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1826-1833 ◽  
Author(s):  
LE Toothaker ◽  
DA Gonzalez ◽  
N Tung ◽  
RS Lemons ◽  
MM Le Beau ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Myosin Heavy Chain ◽  
Cell Lines ◽  
Heavy Chain ◽  
Myeloid Cell ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Granulocytic Differentiation ◽  
Human Leukocytes

Abstract We have isolated 5′ cDNA clones encoding a member of the cellular myosin heavy chain gene family from human leukocytes. The predicted amino acid sequence shows 93% identity to a chicken cellular myosin heavy chain, 76% to chicken smooth muscle, and 40% to human sarcomeric myosin heavy chain. The mRNA is expressed as a 7.4- to 7.9-kb doublet in many nonmuscle cells, and is upregulated in myeloid cell lines on induction from a proliferating to a differentiated state. Antisera raised against a peptide made from the predicted amino acid sequence specifically reacts with a 224-Kd polypeptide in leukocyte cell lines, and the protein is also upregulated during the induction of monocytic and granulocytic differentiation in these cells. The gene for this cellular myosin heavy chain maps to chromosome 22, bands q12.3-q13.1, demonstrating that it is not located in the previously described sarcomeric gene clusters on chromosomes 14 and 17. This cellular myosin heavy chain may be a major contractile protein responsible for movement in myeloid cell lines because no mRNA for sarcomeric myosin heavy chain is detected in these cells.

scholarly journals Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

scholarly journals Cloning and sequencing of protochlorophyllide reductase

1990 ◽  
Vol 265 (3) ◽  
pp. 789-798 ◽  
Author(s):  
P M Darrah ◽  
S A Kay ◽  
G R Teakle ◽  
W T Griffiths
Keyword(s):  
Amino Acid ◽  
Chemical Analysis ◽  
Amino Acid Sequence ◽  
Cdna Library ◽  
Avena Sativa ◽  
Cdna Clones ◽  
Lambda Phage ◽  
Cloning And Sequencing ◽  
Protochlorophyllide Reductase ◽  
Cnbr Cleavage

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a lambda-phage gt11 cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase, p127 codes for more than 95% of the reductase protein.

Purification and structural characterization of ovine placental lactogen

1990 ◽  
Vol 126 (1) ◽  
pp. 141-149 ◽  
Author(s):  
W. C. Warren ◽  
R. Liang ◽  
G. G. Krivi ◽  
N. R. Siegel ◽  
R. V. Anthony
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Structural Characterization ◽  
Binding Sites ◽  
Untranslated Region ◽  
Fetal Liver ◽  
Radioreceptor Assay ◽  
Placental Lactogen ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones

ABSTRACT Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4·2 mg of oPL, with an ∼ 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0·18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4·1 nmol/l) and oPRL (ED50 = 1·1 μmol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a λZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides). The predicted amino acid sequence derived from the nucleotide sequence confirmed homology to bPL (67%) and oPRL (48%). Little amino acid sequence existed with other PLs (≤29%) or GH proteins (≤27%). These results suggest that oPL and oGH are more biologically similar in their ability to compete for fetal liver binding sites, but that oPL is structurally more similar to oPRL. Elucidation of exact structure–function relationships for oPL will, however, require further investigation. Journal of Endocrinology (1990) 126, 141–149

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