Georges Spohr

MEF‐2 function is modified by a novel co‐repressor, MITR

The MEF‐2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types. In skeletal muscle cells, MEF‐2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression. Similar interactions with tissue or lineage‐specific cofactors may also underlie MEF‐2 function in other cell types. In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two‐hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF‐2 interacting transcription repressor (MITR) protein binds to the N‐terminal MADS/MEF‐2 region of the MEF‐2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF‐2‐dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, we propose that MITR acts as a co‐repressor, recruiting a specific deacetylase to downregulate MEF‐2 activity.

Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos

A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.

Organization and sequence analysis of a cluster of repetitive DNA elements from Xenopus laevis

Abstract A genomic DNA fragment of 4.9 × 10 3 base-pairs from Xenopus laevis , which contains transcribed repetitive as well as single copy sequences, has been analyzed in detail by genomic blot, restriction enzyme and sequence analysis. The results reveal the existence of several different repeats, which are intermingled in the examined DNA segment but not always adjacent to each other at other genomic sites. One of the dispersed repetitive elements, which is repeated approximately 100,000 times in the genome, consists of eight tandemly repeated subunits of 77 to 79 base-pairs which show an amazingly high degree of sequence homology. The other repetitive sequences do not show such a regular pattern but contain small groups of nucleotides that are represented several times. Transcripts complementary both to the unique sequences and to the different repeats exist in total oocyte RNA and in RNA from polysomes of stage 40 embryos. While the unique sequence is complementary to only one distinct RNA species, many different RNA molecules carry sequences complementary to both strands of the tandemly repeated repetitive DNA.

Structural analysis of a cDNA clone from Xenopus laevis containing a repetitive sequence element

Abstract Total polysomal RNA from Xenopus laevis stage 40 embryos was probed for the presence of repetitive sequences by Northern blot analysis with a genomic DNA fragment which had previously been shown to contain several repetitive sequence elements ( Spohr et al. , 1981 ). The analysis revealed that various presumptive mRNAs contain sequences complementary to the repetitive probe. Consequently, a cDNA library was constructed and screened with the same probe. Forty-eight positive recombinants containing eucaryotic inserts of 300–700 base pairs were isolated and one such clone was characterized in detail. Analysis of its nucleotide sequence revealed the presence of an open reading frame for 118 amino acids. Comparison of nucleotide sequences located 3′ to this presumptive protein coding region with the sequence of the genomic DNA fragment used as a probe clearly identifies and allows one to define the exact location of the repetitive element in the cloned cDNA. This analysis shows furthermore that one portion of the repeated sequence is highly conserved in the two members of this repetitive sequence family, whereas the other part is more divergent. In this area blocks of oligonucleotides are scattered between nonhomologous DNA stretches. The occurrence frequency of the presumptive mRNAs which carry repetitive elements homologous to the used repetitive probe is suggested to be close to that of rare mRNAs.

Identification of free cytoplasmic globin mRNA of duck erythroblasts by hybridization to anti-messenger DNA and by cell-free protein synthesis.

The cytoplasm of animal cells contains mRNA not associated with ribosomes which exists in the form of ribonucleoprotein (RNP) complexes [ 1,2] . It has been suggested that such complexes represent a pool of mRNA intermediate between the nuclear pre-mRNA and the mRNP complex active in translation [l-4] . We have described in immature duck erythrocytes a cytoplasmic 9 S RNA not associated with ribosomes. This free cytoplasmic 9 S RNA has the same electrophoretic mobility as polyribosoma19 S globin mRNA; its rate of synthesis and decay is consistent with, but not a proof of, a role as a precursor to polyribosomal 9 S mRNA [4] . In this paper we present direct proof for the existence of a cytoplasmic pool of genuine globin mRNA free from the ribosomes (free mRNA) by demonstrating the capacity of the free 9 S RNA to be translated into globins and to hybridize with anti-messenger DNA (amDNA) copied from globin mRNA by reverse transcriptase.

Structural analysis of repetitive sequence elements transcribed in early development of Xenopus laevis

A cDNA library prepared from mRNA ofXenopus laevis embryos was screened with a genomic DNA fragment containing various transcribed repetitive sequence elements.Comparison of the nucleotide sequence of two isolated cDNAs and their genomic relatives allows one to define two transcribed repetitive sequence elements. One of them belongs to a highly reiterated family and consists of a tandem array of homologous subunits of 77–80 bp. The other is reiterated approximately 2 200 times, has a size of 260 bp and displays a conserved region of 135 bp.The data are consistent with the presence of repetitive sequence transcripts in the 3′ part of mRNA molecules.