Transcription initiation: imposing specificity by localization.

Many crucial cellular enzymes--including RNA polymerases, kinases, phosphatases, proteases, acetylaters, etc.--have multiple potential substrates. Regulation entails substrate selection, a process effected by a mechanism we call regulated localization. This formulation is particularly well illustrated by the mechanisms of gene regulation. Analysis of these mechanisms reveals that regulated localization requires simple molecular interactions. These molecular interactions readily lend themselves to combinatorial control. This system of regulation is highly 'evolvable'. Its use accounts, at least in part, for the nature of many of the complexities observed in biological systems.

Imposing specificity by localization: mechanism and evolvability.

Cells detect extracellular signals by allostery and then give those signals meaning by 'regulated localization'. We suggest that this formulation applies to many biological processes and is particularly well illustrated by the mechanisms of gene regulation. Analysis of these mechanisms reveals that regulated localization requires simple molecular interactions that are readily used combinatorially. This system of regulation is highly 'evolvable', and its use accounts, at least in part, for the nature of the complexities observed in biological systems.

Transcriptional activation by recruitment.

The recruitment model for gene activation stipulates that an activator works by bringing the transcriptional machinery to the DNA. Recent experiments in bacteria and yeast indicate that many genes can be activated by this mechanism. These findings have implications for our understanding of the nature of activating regions and their targets, and for the role of histones in gene regulation.

Newt myotubes reenter the cell cycle by phosphorylation of the retinoblastoma protein.

Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb-/- mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467-1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb-/- mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.

Receptor isoform specificity in a cellular response to retinoic acid.

The effects of retinoic acid on cell proliferation, differentiation and patterning are thought to be mediated by the various retinoic acid receptors. Different receptor types are encoded by distinct genes (alpha, beta, and gamma), whereas various isoforms within each type are encoded by splicing variants resulting from the use of alternative promoters. The only region that differs between isoforms is the N-terminal A region containing a transcriptional activating domain. It has been proposed that these alternative A regions confer distinct activities on the receptors, thus allowing each to mediate specific effects of retinoic acid, but it has been difficult to demonstrate such isoform specificity as most cells express a number of different retinoic acid receptors. In an attempt to test whether different isoforms can mediate distinct biological effects we are focusing on retinoic-acid-dependent growth inhibition of newt limb cells. We have constructed chimaeric receptors in which the retinoic acid binding domain of each of five newt retinoic acid receptors has been replaced with a thyroid hormone (T3) binding domain. These constructs were introduced individually into cells whose growth rate was then measured in the presence of T3. The chimaeric alpha 1 receptor mediated T3-dependent inhibition of proliferation that was comparable to that given by retinoic acid, whereas the alpha 2 isoform had no activity in this assay, nor did the delta 1A, delta 1B and Delta 2 receptors. When the A region was deleted from the alpha 1 chimaera it remained a potent T3-dependent transcriptional activator, but no longer mediated T3-dependent growth inhibition. In contrast, when the A region of alpha 1 was transferred to a delta chimaeric receptor, the resulting molecule was fully active in T3-dependent growth inhibition. This is the first direct evidence for isoform specificity in a biological response to retinoic acid, and demonstrates that the specificity of this response is confined to the A region.

Limb development. The budding role of FGF.

Implantation of beads soaked in fibroblast growth factor into the flank of a chick embryo causes extra limbs to be formed, suggesting that FGF is important in initiating limb buds.

Expression and activity of the newt Msx-1 gene in relation to limb regeneration.

The Msx-1 homeobox gene is expressed in various contexts during vertebrate development, including the progress zone of the avian and mouse limb bud. Expression of mouse Msx-1 in a cultured myogenic cell line conferred a transformed phenotype and inhibited fusion into myotubes. It has been proposed that Msx-1 expression is required to maintain certain cells in a proliferating and undifferentiated state and may be associated with the ability to regenerate limbs. Urodele amphibians such as the newt regenerate their limbs by formation of a growth zone or blastema, and we have isolated and sequenced newt Msx-1 (NvMsx-1) from a limb blastemal cDNA library. NvMsx-1 expression was detectable in RNA preparations from both limb and tail and their regeneration blastemas, although cultured cells established from limb blastemal mesenchyme gave negative results. When either COS cells or cultured newt blastemal cells were cotransfected with an expression vector for NvMsx-1 and reporter plasmids containing multiple homeobox protein binding sites, NvMsx-1 repressed reporter expression. If NvMsx-1 was expressed together with a marker enzyme in cultured newt blastemal cells, no significant difference in DNA synthesis was observed relative to control transfectants. When myogenic mononucleate cells were transfected with NvMsx-1 and subsequently exposed to low serum to promote fusion, the fraction of Msx-1 positive cells in myotubes was comparable to a control transfected population analysed in the same culture. These results indicate that although Msx-1 expression could be important for limb regeneration, it does not exert a cell-autonomous effect on proliferation or myogenic differentiation of cultured blastemal cells.

Chimeric retinoic acid/thyroid hormone receptors implicate RAR-alpha 1 as mediating growth inhibition by retinoic acid.

Retinoic acid (RA) affects the growth and differentiation of cells in culture, usually to decrease the growth rate. In amphibian limb regeneration RA has the remarkable ability to affect pattern formation by changing positional identity, but its initial action on the limb is to inhibit division of the blastemal progenitor cells. Newt limb blastemal cells also show this inhibition in culture. In order to investigate the role of different RA receptors (RARs) in the RA response, the hormone binding domain of the newt RARs alpha 1 and delta 1 was replaced with the corresponding region from the Xenopus thyroid hormone receptor-alpha (TR-alpha). In COS cells transfected with each of the chimeras, transcription was activated after exposure to thyroid hormone (T3). Their profile of activity on three different response elements was indicative of RAR specificity and not TR specificity. After transfection of cultured newt blastemal cells with a DNA particle gun, the chimeras were equally active in stimulating T3-dependent transcription of two different synthetic reporter genes. Blastemal cells were transfected with chimeras or control plasmids along with a marker plasmid expressing beta-galactosidase, exposed to RA or T3 and labelled with [3H]thymidine followed by autoradiography. The alpha 1 chimera gave T3-dependent inhibition of growth, comparable to the effect exerted by RA itself, whereas the delta 1 chimera and control plasmids were inactive. The results imply that RAR-alpha 1 mediates the effects of RA on blastemal cell growth.

Activators and targets.

Proteins that activate genes are quite disparate in character; in particular, some work 'universally' and others do not. A simple model can accommodate most of the recently published results.

Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes.

We have identified the recognition sequence for the Citrobacter freundii restriction endonuclease CfrA, a member of the A-family of type I R-M enzymes. This bipartite target sequence differs in both its components from those of other type I enzymes. We determined the nucleotide sequence of its specificity gene (hsdS) and a comparison of this with its relative EcoA identifies two extensive variable regions, an organization analogous to that found in the K-family of type I R-M enzymes. The specificity polypeptides of the A-family, unlike those of K, have an N-terminal conserved region, and this includes a sequence repeated within the central conserved region. A second repeat sequence, identified at the amino acid level, coincides with the only sequence similarity common to all type I S polypeptides. Sequences immediately downstream from the hsdS genes of EcoA, CfrA, EcoK, B and D are almost identical, consistent with an allelic chromosomal location.

Conservation of complex DNA recognition domains between families of restriction enzymes.

One polypeptide, designated S, confers sequence-specificity to the multisubunit type I restriction enzymes. Two families of such enzymes, K and A, include members that recognize diverse, bipartite, target sequences. The S polypeptides of the K family, while having areas of near identity, also contain two extensive regions of variable sequence. We now show that one of these, comprising the N-terminal 150 amino acids, specifies recognition of one component of the bipartite target sequence. We have determined the sequence recognized by EcoE, a member of the A family. This sequence, 5'GAG(N7)ATGC, has the trinucleotide GAG in common with EcoA and with StySB of the K family. We determined the nucleotide sequences of the S genes of EcoA and EcoE, and compared their predicted amino acid sequences with each other and with those of the five members of the K family. There is no general sequence similarity between families, but the domain of the S polypeptide of StySB, which specifies GAG, shows nearly 50 per cent identity with the amino variable region of the S polypeptides of EcoA and EcoE. A complex domain that recognizes and directs methylation of GAG is therefore common to enzymes of generally dissimilar amino acid sequence.

Reassortment of DNA recognition domains and the evolution of new specificities.

Type I restriction enzymes comprise three subunits only one of which, the S polypeptide, dictates the specificity of the DNA sequence recognized. Recombination between two different hsdS genes, SP and SB, led to the isolation of a system, SQ, which had a different specificity from that of either parent. The finding that the nucleotide sequence recognized by SQ is a hybrid containing components from both the SP and SB target sequences suggested that DNA recognition is carried out by two separable domains within each specificity polypeptide. To test this we have made the recombinant gene of reciprocal structure and demonstrate that it encodes a polypeptide whose recognition sequence, deduced in vivo, is as predicted by this model. We also report the sequence of the SB specificity gene, so that information is now available for the five known members of this family of enzymes. All show a similar organization of conserved and variable regions. Comparisons of the predicted amino acid sequences reveal large non-conserved areas which may not even be structurally similar. This is remarkable since these different S subunits are functionally identical, except for the specificity with respect to the DNA sequence with which they interact. We discuss the correlation of the variation in polypeptide sequence with recognition specificities.

Abortive infection by bacteriophage Me1 of Escherichia coli K12 strains bearing the plasmid ColV, I-K94.

Bacteriophage Me1 is unable to grow on Escherichia coli strains harbouring the ColV,I-K94 plasmid. The nature of this inhibition was investigated, and it was found not to be due to restriction, superinfection exclusion or receptor-mediated resistance, but to be a new example of plasmid-mediated abortive infection. Investigation of events occurring during abortive Me1 infection revealed some differences from previously described cases, especially with regard to late protein synthesis, which did occur, albeit showing abnormal amounts of some proteins. No major differences were observed in membrane permeability of productively and abortively infected cells. Phage-directed DNA synthesis was reduced in abortively infected cells. Comparative studies of Me1 and T4 revealed a striking similarity despite some minor differences.