Abstract
The protein of Myc modulator 1 (Mm-1) has been reported to repress the transcriptional activity of the proto-oncogene c-Myc in humans. Moreover, it was shown to be the subunit 5 of human prefoldin (PFD). So far, this gene and its homologs have been isolated and sequenced in many organisms, such as mammals and fish, but has not been sequenced for any amphibian or reptile. In order to better understand the function and evolution of Mm-1, we isolated a full-length Mm-1 cDNA (BgMm-1, GenBank accession no. EF211947) from Bufo gargarizans (Cantor, 1842) using RACE (rapid amplification of cDNA ends) methods. Mm-1 in B. gargarizans is 755 bp long, comprising an open reading frame (ORF) of 459 bp encoding 152 amino acids. The amino acid sequence had a prefoldin α-like domain, partially including a typical putative leucine zipper motif. BgMm-1 showed high similarity to its homolog of Mus musculus Linnaeus, 1758 (82%) and Homo sapiens Linnaeus, 1758 MM-1 isoform a (81%) at the amino acid level. The protein secondary structure modeled with the SWISS MODEL server revealed that there were two α-helices and four b-strands in BgMm-1 as its human orthologue, and both proteins belonged to the a class of PFD family. The phylogenetic relationships of Mm-1s from lower archaea to high mammals was consistent with the evolution of species, meanwhile the cluster result was consistent with the multiple alignment and the sequence identity analysis. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis demonstrated that BgMm-1 expressed widely in ten tissues of adult toad. These results can be helpful for the further investigation on the evolution of Mm-1.
Highlights
Myc modulator 1 (Mm-1), a novel c-Myc-binding protein, was first isolated using the yeast two-hybrid screening of a human Hela cell cDNA library (MORI et al 1998)
The protein secondary structure modeled with the SWISS MODEL server revealed that there were two ␣-helices and four -strands in BgMm-1 as its human orthologue, and both proteins belonged to the ␣ class of PFD family
FUJIOKA et al (2001) confirmed that Mm-1 was a candidate for a tumor suppressor in leukemia/lymphoma and tongue cancer that controls the transcriptional activity of c-Myc
Summary
A male and a female of B. gargarizans ( termed Chinese large toad) were obtained from the Wuhu city in Anhui province, China. To amplify a conservative fragment of BgMm1 cDNA, a pair of degenerate primers was designed based on the conserved amino acid sequences that have been found in the alignment of mammal and fish Mm-1s (mF: 5’-CTG(A/C)A(A/ G/T)GTCGT(G/C/T)CA(A/G)(A/G)CCAA(A/G/C)(T/C)-3’ and mR:5’-NTGTGCC(A/G)GCCTG(C/A/T)GCCG(C/T)-3’). Aligned amino acid sequences were used to construct a phylogenetic tree by the neighbor-joining method using MEGA V3.0 program. Total RNA samples were isolated from eleven tissues of adult B. gargarizans, such as muscle, heart, brain, kidney, spleen, testis, ovary, lung, liver, pancreas and stomach, with TRIzol (Invitrogen) according to the manufacturer’s instructions. Single-stranded cDNA were synthesized from 5 μg of total RNA by reverse transcription with M-MLV RTase (TaKaRa, Dalian, China), primed with Oligo(dT), and used as template for PCR. The PCR products were detected on 1.5% agarose gels
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