Transcription of the Agrobacterium Ti plasmid in the bacterium and in Crown Gall tumors

Regions of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid which are transcribed in the bacterium or in two tobacco Crown Gall tumors were localized. Complementary DNA (cDNA) probes made to bacterial or tumor RNA were hybridized to blots of the Ti-plasmid or cloned “T”-DNA restriction endonuclease fragments digested with various restriction endonucleases. Extensive regions of the Ti plasmid are transcribed in the bacterium grown in minimal or rich medium. An additional region of the plasmid, which has previously been defined genetically as coding for proteins responsible for octopine utilization and conjugative T-plasmid transfer, is transcribed when the bacteria are induced with octopine. This region is transcribed constitutively in a mutant which is constitutive for octopine utilization. Another additional region of the plasmid is transcribed when the bacteria are induced with agropine. All sections of the “T” DNA are weakly transcribed in the bacterium. In contrast to this, specific regions of the “T” DNA are transcribed into both polyadenylated and nonpolyadenylated RNA in the tumors. The selectivity with which regions are transcribed in the tumor may indicate that the “T” DNA has “evolved” for best use in a eucaryotic cell.     

Why do primordial germ cells migrate through an embryo and what does it mean for biological evolution?

An explanation of the role of primordial germ cell (PGC) migration during embryogenesis is proposed. According to the hypothesis, various PGCs during their migrations through an early embryo are contacting with anlagen of organs and acquiring nonidentical organ specificities. An individual PGC gets such an organ specificity, which corresponds to specificity of the first anlage with which this PGC has the first contact. As a result, the cellular descendants of PGCs (oocytes or spermatocytes) will express nonidentical organ-specific receptors, hence becoming functionally heterogeneous. Therefore, each clone of germ cells becomes capable of recognizing specifically the molecular signals that correspond only to “its” organ of the body. Such signals are produced by the body’s organ when it functions in an extreme mode. Signals from the “exercising” organ of the body are delivered to the gonad only via the brain retransmitter, which is composed of neurons grouped as virtual organs of a homunculus. Homunculi are so-called somatotopic maps of the skeletomotor and other parts of the body represented in the brain. Signals, as complexes of regulatory RNAs and proteins, are transported from the “exercising” organ of the body to the corresponding virtual organ of the homunculus where they are processed and then forwarded to the gonad. The organ-specific signal will be selectively recognized by certain gametocytes according to their organ specificity, and then it will initiate the directed epimutation in the gametocyte genome. The nonrandomness of the gene order in chromosomes, that is the synteny and genetic map, is controlled by the so-called creatron that consolidates the soma and germline into a united system, providing the possibility of evolutionary responses of an organism to environmental influences.     

On the origin and evolution of the genetic code. II. Origin of the genetic code as a primordial collector language. The pairing-release hypothesis.

The genetic code is treated as a language used by primordial “collector societies” of tRNA molecules (meaning: societies of RNA molecules specialized in the collection of amino acids and possibly other molecular objects), as a means to organize the delivery of collected material. Its origin is ascribed to the utilization of the complementarity between each tRNA and the genome segment from which it was originally copied, as a means to identify by annealing operations the tRNA molecules returning from their collection trips, and elicit the release of the amino acids they are carrying (the pairing release hypothesis).The gradual conversion of tRNA complements into codon-triplets in the regions of the primordial RNA genomes which specialized in the task of directing the delivery of amino acids by returning tRNA molecules, is ascribed to the removal of genetic redundancy in a gradual reorganization process.A reconstruction of the codon-triplets in one of the earliest genetic codes is attempted by the wobbling reintroduction procedure used in a preceding paper.     

Selection of repeated sequences of homologous and heterologous DNA during in vitro transcription by maize RNA polymerase

RNA synthesized $\text{in}\text{vitro}$ by maize RNA polymerase II arises in part from repeated DNA sequences, since significant hybridization to the parent DNA occurs with low concentrations of RNA and DNA. Over three times as much “repeated sequence” RNA is transcribed from maize as from calf thymus DNA.     

Proteins, exons and molecular evolution

The discovery of the eukaryotic gene structure has prompted research into the potential relationship between protein structure and function and the corresponding exon/intron patterns. The exon shuffling hypothesis put forward by Gilbert and Blake suggests the encodement of structural and functional protein elements by exons which can recombine to create novel proteins. This provides an explanation for the relatively rapid evolution of proteins from a few primordial molecules. As the number of gene and protein structures increases, evidence of exon shuffling is becoming more apparent and examples are presented both from modern multi-domain proteins and ancient proteins. Recent work into the chemical properties and catalytic functions of RNA have led to hypotheses based upon the early existence of RNA. These theories suggest that the split gene structure originated in the primordial soup as a result of random RNA synthesis. Stable regions of RNA, or exons, were utilised as primitive enzymes. In response to selective pressures for information storage, the activity was directly transferred from the RNA enzymes or ribozymes, to proteins. These short polypeptides fused together to create larger proteins with a wide range of functions. Recent research into RNA processing and exon size, discussed in this review, provides a clearer insight into the evolutionary development of the gene and protein structure.     

The gene and messenger RNA for adenovirus polypeptide IX

A small RNA species, distinct from the VA RNAs, has been identified in HeLa cells infected with adenovirus type 2. The RNA, which has been purified using a novel screening procedure, is polyadenylated, sediments at 9S and has an estimated length of 550 nucleotides. In a cell-free translation system, the 9S RNA directs the synthesis of virion polypeptide IX, molecular weight 12,000 daltons. The location of its gene has been established by hybridization of the RNA to fragments of viral DNA produced by cleavage with restriction endonucleases: it spans position 10.0 on the r strand of the viral genome. These results unexpectedly place the gene for a “late” protein within a region of the genome which is transcribed early during infection.     

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.     

Changes in "template-bound" and "free" RNA polymerase activities in isolated nuclei from Xenopus laevis embryos

Nuclei have been isolated from Xenopus laevis embryos and incubated under conditions allowing RNA synthesis to proceed for more than 3 h. The RNA molecules synthesized on the endogenous template are stable, heterogeneous in size and correspond to the activities of the three RNA polymerases.In these in vitro conditions we have determined the extent of activity of the three RNA polymerases during the embryonic development from blastula to swimming tadpole. Our results on isolated nuclei are in good agreement with the changes in RNA synthesis which take place during normal embryonic development.We have measured both the “template-bound” and the “free” activities of each of the three RNA polymerases during development. Amongst the total RNA polymerase activities engaged on the template, the proportion of polymerase I increases as development proceeds: at the blastula stage, there is practically no RNA polymerase I engaged on the template, whereas in swimming tadpoles, RNA polymerase I amounts to about 90% of the RNA polymerases bound to the DNA. Conversely, RNA polymerase I represents the major part of free RNA polymerases in blastula nuclei.Autoradiography of incubated nuclei shows that, at least in swimming tadpoles nuclei, both “free” and “template-bound” RNA polymerase I are localized in the nucleoli.The evolution of “template-bound” RNA polymerase II activity during development is quite different from that of RNA polymerase I: RNA polymerase II activity represents 75% of engaged polymerase activity in blastulae and only 47% at the swimming tadpoles stage.The results suggest that part of the “free” RNA polymerase I activity might progressively become “template-bound” during embryogenesis.