Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases.

We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain. The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1. Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2-associated protein but not yet shown to have Met-AP activity. Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12-fold increases in Met-AP activity on different peptide substrates. The epitope-tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity. To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains. The map2 null strain, like the map1 null strain, is viable but with a slower growth rate. The map1, map2 double-null strains are nonviable. Thus, removal of N-terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone.

Artificial regulation of gene expression in Escherichia coli by RNase P.

Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.

Identification and characterization of putative transposable DNA elements in solanaceous plants and Caenorhabditis elegans.

Several families of putative transposable elements (TrEs) in both solanaceous plants and Caenorhabditis elegans have been identified by screening the DNA data base for inverted repeated domains present in multiple copies in the genome. The elements are localized within intron and flanking regions of many genes. These elements consist of two inverted repeats flanking sequences ranging from 5 bp to > 500 bp. Identification of multiple elements in which sequence conservation includes both the flanking and internal regions implies that these TrEs are capable of duplicative transposition. Two of the elements were identified in promoter regions of the tomato (Lycoperiscon esculentum) polygalacturonase and potato (Solanum tuberosum) Win1 genes. The element in the polygalacturonase promoter spans a known regulatory region. In both cases, ancestral DNA sequences, which represent potential recombination target sequences prior to insertion of the elements, have been cloned from related species. The sequences of the inverted repeated domains in plants and C. elegans show a high degree of phylogenetic conservation. While frequency of the different elements is variable, some are present in very high copy number. A member of a single C. elegans TrE family is observed approximately once every 20 kb in the genome. The abundance of the described TrEs suggests utility in the genomic analysis of these and related organisms.

A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.

Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.

To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli.

Plasmid-encoded addiction genes augment the apparent stability of various low copy number bacterial plasmids by selectively killing plasmid-free (cured) segregants or their progeny. The addiction module of plasmid prophage P1 consists of a pair of genes called phd and doc. Phd serves to prevent host death when the prophage is retained and, should retention mechanisms fail, Doc causes death on curing. Doc acts as a cell toxin to which Phd is an antidote. In this study we show that host mutants with defects in either subunit of the ClpXP protease survive the loss of a plasmid that contains a P1 addiction module. The small antidote protein Phd is fully stable in these two mutant hosts, whereas it is labile in a wild-type host. We conclude that the role of ClpXP in the addiction mechanism of P1 is to degrade the Phd protein. This conclusion situates P1 among plasmids that elicit severe withdrawal symptoms and are able to do so because they encode both a cell toxin and an actively degraded macromolecule that blocks the synthesis or function of the toxin.

Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America.

The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote street rabies virus (COSRV) revealed that there are unique features associated with SHBRV. In vitro studies showed that, while the susceptibility of neuroblastoma cells to infection by both viruses was similar, the infectivity of SHBRV was much higher than that of COSRV in fibroblasts (BHK-21) and epithelial cells (MA-104), particularly when these cells were kept at 34 degrees C. At this temperature, low pH-dependent fusion and cell-to-cell spread of virus is seen in BHK-21 cells infected with SHBRV but not with COSRV. It appears that SHBRV may possess an unique cellular tropism and the ability to replicate at lower temperature, allowing a more effective local replication in the dermis. This hypothesis is supported by in vivo results which showed that while SHBRV is less neurovirulent than COSRV when administered via the intramuscular or intranasal routes, both viruses are equally neuroinvasive if injected intracranially or intradermally. Consistent with the above findings, the amino acid sequences of the glycoproteins of SHBRV and COSRV were found to have substantial differences, particularly in the region that contains the putative toxic loop, which are reflected in marked differences in their antigenic composition. Nevertheless, an experimental rabies vaccine based on the Pittman Moore vaccine strain protected mice equally well from lethal doses of SHBRV and COSRV, suggesting that currently used vaccines should be effective in the postexposure prophylaxis of rabies due to SHBRV.

Human TOP3: a single-copy gene encoding DNA topoisomerase III.

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.

A splice variant of alpha 6 integrin is associated with malignant conversion in mouse skin tumorigenesis.

The epithelial-specific integrin alpha 6 beta 4 is suprabasally expressed in benign skin tumors (papillomas) and is diffusely expressed in carcinomas associated with an increase in the proliferating compartment. Analysis of RNA samples by reverse transcriptase-PCR and DNA sequencing revealed that chemically or oncogenically induced papillomas (n = 8) expressed a single transcript of the alpha 6 subunit, identified as the alpha 6 A splice variant. In contrast, carcinomas (n = 13) expressed both alpha 6A and an alternatively spliced form, alpha 6B. Primary keratinocytes and a number of keratinocyte cell lines that vary in biological potential from normal skin, to benign papillomas, to well-differentiated slowly growing carcinomas exclusively expressed alpha 6A. However, I7, an oncogene-induced cell line that produces highly invasive carcinomas, expressed both alpha 6A and alpha 6B transcript and protein. The expression of alpha 6B in I7 cells was associated with increased attachment to a laminin matrix compared to cell lines exclusively expressing alpha 6A. Furthermore, introduction of an alpha 6B expression vector into a papilloma cell line expressing alpha 6A increased laminin attachment. When a papilloma cell line was converted to an invasive carcinoma by introduction of the v-fos oncogene, the malignant cells expressed both alpha 6A and alpha 6B, while the parent cell line and cells transduced with v-jun or c-myc, which retained the papilloma phenotype, expressed only alpha 6A. Comparative analysis of alpha 6B expression in cell lines and their derived tumors indicate that alpha 6B transcripts are more abundant in tumors than cell lines, and alpha 6B is expressed to a greater extent in poorly differentiated tumors. These results establish a link between malignant conversion and invasion of squamous tumor cells and the regulation of transcript processing of the alpha 6 beta 4 integrin.