Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III.

Purified RNA polymerase II (pol II) from the yeast Saccharomyces cerevisiae pauses without releasing at many locations during in vitro transcription. Pausing can be induced by intrinsic DNA sequence as well as by specific DNA bound proteins such as the RNA pol I termination factor, Reb1p, or lac repressor. Addition of rho termination factor from E. coli induces RNA pol II to release at all of these pause sites. Rho-induced release of pol II requires both a rho binding site in the transcript upstream of the pause sites as well as hydrolysis of ATP. In contrast, rho factor has no effect on either pausing or release by RNA pol I or III. When combined with previous observations, these results suggest that RNA pol II may terminate by a mechanism closely related to the rho-dependent mechanism of prokaryotes. In contrast, pol I and III appear to utilize a mechanism more related to the rho-independent terminators of prokaryotes.

Terminating transcription in eukaryotes: lessons learned from RNA polymerase I.

Within the past few years, the genes encoding transcription terminator proteins for RNA polymerase I (pol I) have been cloned from organisms as diverse as yeast and mammals. The availability of terminator proteins has allowed construction of in vitro transcription systems that terminate pol I at the same sites as used in vivo and thus allows study of termination mechanisms. This has resulted in a burst of information concerning pol I termination mechanisms, which this review will attempt to summarize.

The yeast transcription terminator for RNA polymerase I is designed to prevent polymerase slippage.

A transcription terminator for RNA polymerase I (polI) in the yeast, Saccharomyces cerevisiae, is composed of two essential elements, the 11bp binding site for Reb1p and an upstream T-rich element coding for the last 10-12 nucleotides of the terminated transcript. We now show that, if the upstream element is changed to homopolymer T residues, polI undergoes iterative slippage, long poly(U) tails are added to the transcript, and termination is impaired. Reinsertion of one or two non-T residues within a critical region prevents iterative slippage and reinstates termination. A survey of naturally occurring terminators reveals that many contain T-rich upstream regions with non-T residues situated appropriately to prevent slippage. We discuss the possibility that the first step in slippage, backward sliding of both the transcript and the catalytic center of the polymerase, may be an obligatory step in the normal termination process.

The release element of the yeast polymerase I transcription terminator can function independently of Reb1p.

The Saccharomyces cerevisiae polymerase I (polI) transcription terminator utilizes a DNA-binding protein (Reb1p) as part of a signal that causes the polymerase to pause prior to release from the template. To study the release element of the terminator, independent of the Reb1p pause signal, we have replaced the Reb1p binding site with the binding site for the lac repressor, which acts as a self-contained heterologous pause signal for polI. Release efficiency is maximal when the lac repressor causes polI to pause in exactly the same position that Reb1p would have caused it to pause, suggesting that polI must be precisely positioned for transcript release to occur. Mutational analysis shows that the release element is a region rich in T residues which codes for the extreme 3' end of the transcript and which has no apparent ability to form hairpins when transcribed into RNA. We discuss possible mechanisms whereby this polI release element might function.

Transcription termination of RNA polymerase I due to a T-rich element interacting with Reb1p.

All transcription terminators for RNA polymerase I (pol I) that have been studied so far, ranging from yeast to humans, require a specific DNA binding protein to cause termination. In yeast, this terminator protein has been identified as Reb1p. We now show that, in addition to the binding site for Reb1p, the yeast pol I terminator also requires the presence of a T-rich region coding for the last 12 nucleotides of the transcript. Reb1p cooperates with this T-rich element, both to pause the polymerase and to effect release of the transcript. These findings have implications for the termination mechanism used by all three nuclear RNA polymerases, since all three are known to pause at this terminator.

A model for transcription termination by RNA polymerase I.

The transcription termination site for yeast RNA polymerase I requires not only an 11 bp binding site for Reb1p, but also about 46 bp of 5' flanking sequence. We propose that Reb1p bound to its site is part of a pause element, while the 5' flanking sequence contains a release element. Pausing requires little other than the DNA-binding domain of Reb1p and is not specific for polymerase I. The release element, however, can be polymerase specific. We propose a general model for eukaryotic transcription terminators in which termination occurs when a relatively nonspecific signal induces polymerase to pause in the context of a release element.

The REB1 site is an essential component of a terminator for RNA polymerase I in Saccharomyces cerevisiae.

We have identified a terminator for transcription by RNA polymerase I in the genes coding for rRNA of the yeast Saccharomyces cerevisiae. The terminator is located 108 bp downstream of the 3' end of the mature 25S rRNA and shares several characteristics with previously studied polymerase I terminators in the vertebrates. For example, the yeast terminator is orientation dependent, is inhibited by its own sequence, and forms RNA 3' ends 17 +/- 2 bp upstream of an essential protein binding site. The recognition sequence for binding of the previously cloned REB1 protein (Q. Ju, B. E. Morrow, and J. R. Warner, Mol. Cell. Biol. 10:5226-5234, 1990) is an essential component of the terminator. In addition, the efficiency of termination depends upon sequence context extending at least 12 bp upstream of the REB1 site.

Cloning and sequencing of Octopus dofleini hemocyanin cDNA: derived sequences of functional units Ode and Odf.

A number of additional cDNA clones coding for portions of the very large polypeptide chain of Octopus dofleini hemocyanin were isolated and sequenced. These data reveal two very similar coding sequences, which we have denoted "A-type" and "G-type." We have obtained complete A-type sequences coding for functional units Ode and Odf; consequently a total of three such unit sequences are now known from a single subunit of one molluscan hemocyanin. This presents the opportunity to make sequence comparisons within one hemocyanin subunit. Domains within one subunit show on the average 42% identity in amino acid residues; corresponding functional units from hemocyanins of different species show degrees of identity of 53-75%. Therefore, molluscan hemocyanins already existed before the individual molluscan classes diverged in the early Cambrian. Sequence comparisons of molluscan hemocyanins with arthropodan hemocyanins and tyrosinases allow us to identify the ligands of the "Copper B" site with high probability. Possible ligands for the "Copper A" site are proposed, based on sequence comparisons between molluscan hemocyanins and tyrosinases. Besides two histidine side chains, a methionine side chain might be involved in binding of Copper A, a result not in conflict with spectroscopic studies.

cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain.

A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified.

[Differential diagnostic studies using the dermatoscope in selected diseases]. / Differentialdiagnositsche Untersuchungen mit dem Dermatoskop bei ausgewählten Erkrankungen.

The Zeis dermatoscope can be used beyond its known applications for precise and detailed skin surface examinations of different types of dermatosis. Differential diagnostic problems are solved by comparative observation of efflorescence and efflorescence fields of similar appearance. The examination series presented, the results of which are discussed in detail, is concerned mainly with surface examinations of parapsoriasis en plaques, seborrhea en plaques and the clinically similar early forms of mycosis fungoides. Also mentioned are examinations of lichen sclerosus et atrophicus and circumscribed scleroderma, lichen planus, Hutchinson-Dubreuilh freckle and similarly appearing facial verruca seborrhoica, as well as fresh dispersions of foreign bodies. The didactic value is underlined by color photographs taken of diseased skin and nails with the dermatoscope at various magnifications. Finally, a description is given of possible instrument extensions.

The larval development of Cliban vittatus (Bosc) (Crustacea: Decapoda; Diogenidae) reared in the laboratory.

1. A table of previous descriptions of larvae of Diogenidae is given. 2. Larvae of the hermit crab Clibanarius vittatus were reared on a diet of Artemia nauplii. Five, rarely four, zoeal stages and megalopa were obtained. Duration, mortality and sizes of larvae from two hatches are listed. 3. Detailed descriptions and figures for each larval stage are presented.

[A new ceiling microscope suspension for plastic and reconstructive microsurgery]. / Ein neues Deckenstativ für die plastische und rekonstruktive Mikrochirurgie.

Since 1969, 168 microsurgical interfascicular nerve transplantations have been performed in the Department for Hand Surgery and Plastic Surgery in the Surgical Clinic of the University of Erlangen. Since the end of 1974, the Zeiss Operation Microscope OPMI 2 has been hung from an electrohydraulic ceiling suspension and has proved invaluable. The construction and function of the apparatus is described. Such suspension of the microscope in combination with original Hanau operation lights, facilitates microsurgery in hand and plastic surgical procedure by saving space, reducing operating time and providing a better opportunity of ensuring sterility in the operating field.