Deletion mutagenesis in M13 by polymerase chain reaction using universal sequencing primers.

A simple procedure is described for the efficient deletion of large DNA sequences. The method involves a combination of oligonucleotide-directed mutagenesis in bacteriophage M13 and amplification of the mutagenized product by polymerase chain reaction. In contrast to other protocols employing polymerase chain reaction, synthesis of only one specific primer is required.

Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica.

cDNA libraries were constructed from pathogenic (HM-1:IMSS) and nonpathogenic (SAW 1734) isolates of Entamoeba histolytica. A cDNA clone (cEH-P1) specific for pathogenic amoebae was identified by screening with a pool of sera from patients with invasive amoebiasis that had been absorbed with nonpathogenic amoebae. This clone was used for the identification of a homologous clone (cEH-NP1) in the cDNA from nonpathogenic amoebae.

Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity.

A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts.

We have isolated the cDNA encoding a novel human myogenic factor, Myf-5, by weak cross-hydridization to the mouse MyoD1 probe. Nucleotide sequence analysis and the identification of the corresponding gene indicate that human Myf-5 is a member of a small gene family which also contains the human homologue to MyoD1. Although structurally related to the mouse factor, the human Myf-5 constitutes a different protein which nevertheless is capable of inducing the myogenic phenotype in embryonic C3H mouse 10T1/2 'fibroblasts'.

The interaction of nuclear proteins with essential promoter element of the chicken cardiac myosin light chain 2 gene is involved in muscle-specific transcription.

A quantitative microinjection procedure has been developed to demonstrate muscle-specific transcription of the myosin light chain 2-A (MLC2-A) promoter in differentiated chicken primary breast muscle cells. Nuclear protein binds to the distal region of the required promoter sequence but not to a mutated version of this sequence. The functional significance of this specific DNA-protein interaction for the promoter activity is demonstrated by 'in vivo' competition of microinjected MLC-CAT reporter construct together with excess of synthetic oligonucleotides encompassing the protein binding sites.

The promoter of the chicken cardiac myosin light chain 2 gene shows cell-specific expression in transfected primary cultures of chicken muscle.

Transcriptional regulation of the chicken cardiac myosin light chain 2 (MLC2-A) gene was investigated in chicken primary myoblast and fibroblast cultures transfected with vector constructs containing the bacterial marker gene for chloramphenicol acetyltransferase (CAT) under the control of the MLC2-A promoter. We here demonstrate that sequences close to the TATA box are sufficient to direct muscle specific and regulated expression of the MLC2-A mRNA. Transcription from MLC2-A promoter/CAT hybrids in myocytes starts from the authentic cap site that is also used in vivo.