Structure and function of the HOX A1 human homeobox gene cDNA.

Homeobox genes code for transcription factors and are arranged in clusters, named A, B, C and D, found on four separate chromosomes in vertebrates. They contain a homeobox DNA sequence which codes for the homeodomain, a region of amino acids responsible for the DNA binding exhibited by these proteins. During embryonic development, the homeobox genes are both spatially and temporally regulated. In teratocarcinoma cell cultures, homeobox genes are regulated by retinoic acid (RA). The cDNAs from the first gene in the human HOX A cluster, HOX A1 (1.6), were cloned and the nucleotide sequence of a full-length cDNA was determined. It is highly homologous to its murine counterpart. Another HOX A1 cDNA was cloned, corresponding to an alternatively spliced form. In vitro translation of the full-length cDNA clone gave rise to a protein of 36 kDa. In PA-1 human teratocarcinoma cells HOX A1 is the earliest HOX A gene to be expressed after treatment with RA. To test whether HOX A1 could function as a early regulator of other HOX A cluster genes, we cotransfected into PA-1 human teratocarcinoma cells sense and antisense HOX A1 cDNAs expressed from an SV40 promoter with a 5.4-kb RA-sensitive HOX A4 (1.4) promoter-cat reporter. We found no effect of HOX A1 on the HOX A4 promoter. However, cotransfection of HOX A5 (1.3) was able to inhibit the HOX A4 promoter activity.

Identification of a retinoic acid-inducible endogenous retroviral transcript in the human teratocarcinoma-derived cell line PA-1.

Retinoic acid (RA), a developmental morphogen, causes activation of a transcript of an endogenous retrovirus-related element in the human teratocarcinoma-derived cell line PA-1. This provirus is defective, and the provirus-related sequences exist as multicopy elements (more than 20 copies) in human DNA. This is the first human endogenous retroviral mRNA that is known to be transcriptionally activated by RA. The nucleotide sequence of the 3,357 bp of this viral cDNA was determined and shows a strong homology to the type C-related human endogenous retroviral proviruses ERV3 and 4-1. This cDNA contains 'R-U5-delta pol-env-U3-R sequences of the provirus. Adjacent to the putative 5' long terminal repeat of this provirus there is an 18-bp sequence complementary to the 3' end of isoleucine tRNA. We named this RA-responsive virus RRHERV-I.

Tumorigenic transformation of spontaneously immortalized fibroblasts from patients with a familial cancer syndrome.

Immortal cell lines arose spontaneously during in vitro culture of initially normal fibroblasts, MDAH041 and MDAH087, from patients with Li-Fraumeni familial cancer syndrome. Fibroblasts from a control donor, MDAH170, maintained a normal morphology and senesced at 31 population doublings. The immortal fibroblasts have several properties of transformed cells. In addition to having acquired an altered morphology and chromosomal anomalies, MDAH041 and MDAH087 have escaped from senescence, growing beyond 300 and 100 population doublings (pd), respectively. As early as 50 pd, these cells can be transformed by an activated H-ras oncogene to form tumors in nude mice. However, MDAH041 immortal cells were resistant to tumorigenic transformation by transfection with the v-abl oncogene.

Low affinity binding sites for 1,4-dihydropyridines in mitochondria and in guinea pig ventricular membranes.

In this paper, we describe the occurrence of both high and low affinity sites for dihydropyridines in crude membrane preparations from guinea pig ventricular tissue. The physiological significance of the low affinity site (apparent dissociation constant = 76 +/- 9 nM) is not currently known; it has, however, a binding capacity which was 300-1000 times that of the high affinity site and was resistant to heat denaturation. The magnitude of the binding to the low affinity site was affected by both the ionic strength of the medium and by the presence of divalent ions. Both unlabeled nitrendipine and nimodipine inhibited [3H]nitrendipine binding at both sites, but verapamil and diltiazem only affected binding at the high affinity site. We also characterized, both kinetically and by equilibrium binding, a low affinity, heat-stable nitrendipine binding site in purified mitochondria. The Bmax for this site was also dependent on ionic strength. This suggests the possibility that the low affinity site in crude membranes is due to mitochondrial contaminants and hence not directly related to voltage-dependent calcium channels.

Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by alpha-neurotoxin cross-linking.

[3H]Methyl-alpha-neurotoxin prereacted with dithiobis(succinimidyl propionate) (DTSP) can be covalently linked to each of the subunits of the nicotinic acetylcholine receptor in membranes from the electric tissue of Torpedo californica. Pronounced changes in the cross-linking pattern are observed upon prior incubation with receptor specific ligands and upon reduction and/or alkylation of the receptor. d-Tubocurarine has been shown to bind to two different sites in receptor-rich membranes. These sites are present in equal numbers but have different affinities [Neubig, R. R., & Cohen, J. B. (1979) Biochemistry 18, 5464-5475; Sine, S., & Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699]. Using d-tubocurarine inhibition of [3H]-methyl-alpha-neurotoxin binding, we demonstrate two inhibitory constants for d-tubocurarine of 67 +/- 21 nM and 4.9 +/- 1.7 microM in unreduced membranes. We utilize the large difference in Ki's to preferentially block toxin cross-linking at the high affinity site for d-tubocurarine. Low concentrations of this competitive antagonist selectively block the cross-linking of toxin to the beta and gamma subunits of the receptor, suggesting that these subunits are located close to the toxin binding site which is also the high-affinity binding site for d-tubocurarine. Reduction of disulfide bonds alters the affinity of the receptor for alpha-neurotoxin. Alterations are also seen in the cross-linking pattern of DTSP-activated [3H]methyl-alpha-neurotoxin to reduced and alkylated membranes in the presence of tubocurarine. The constants for d-tubocurarine inhibition of [3H]methyl-alpha-neurotoxin binding to reduced and alkylated membranes are 172 +/- 52 nM and 2.4 +/- 0.4 microM. The effects of bromoacetylcholine, carbamoylcholine, gallamine, and procaine on the cross-linking pattern are also examined. Our observations are consistent with an arrangement of the subunits in the membrane of alpha beta alpha gamma delta.