Variants in Apaf-1 segregating with major depression promote apoptosome function.

APAF1, encoding the protein apoptosis protease activating factor 1 (Apaf-1), has recently been established as a chromosome 12 gene conferring predisposition to major depression in humans. The molecular phenotypes of Apaf-1 variants were determined by in vitro reconstruction of the apoptosome complex in which Apaf-1 activates caspase 9 and thus initiates a cascade of proteolytic events leading to apoptotic destruction of the cell. Cellular phenotypes were measured using a yeast heterologous expression assay in which human Apaf-1 and other proteins necessary to constitute a functional apoptotic pathway were overexpressed. Apaf-1 variants encoded by APAF1 alleles that segregate with major depression in families linked to chromosome 12 shared a common gain-of-function phenotype in both assay systems. In contrast, other Apaf-1 variants showed neutral or loss-of-function phenotypes. The depression-associated alleles thus have a common phenotype that is distinct from that of non-associated variants. This result suggests an etiologic role for enhanced apoptosis in major depression.

Clinical application of pharmacogenetics.

Pharmacogenetics encompasses the involvement of genes in an individual's response to drugs. As such, the field covers a vast area including basic drug discovery research, the genetic basis of pharmacokinetics and pharmacodynamics, new drug development, patient genetic testing and clinical patient management. Ultimately, the goal of pharmacogenetics is to predict a patient's genetic response to a specific drug as a means of delivering the best possible medical treatment. By predicting the drug response of an individual, it will be possible to increase the success of therapies and reduce the incidence of adverse side effects.

Pharmacogenetics and antiepileptic drugs.

Responses among patients to antiepileptic drugs (AEDs) may be highly variable, with respect to both drug efficacy and safety. Pharmacogenetics addresses the genetic component of such patient variability. Differential response to phenytoin, for example, is related to interindividual genetic differences in the metabolic enzyme CYP2C9, and to a lesser extent, CYP2C19. However, no other AED responses have yet been linked conclusively to specific genes. Further understanding of the role of genes in AED response will depend on clinical investigations coupled with new information and technologies derived from the Human Genome Project. Once the DNA sequences involved in specific AED responses are understood, they can be used as the basis of clinical assays to predict the most likely response in each individual patient. The combination of clinical investigations, genomics, and emerging testing methodologies should lead to new tools for the effective management of epilepsy.

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.

Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA.

The termini of macronuclear DNA molecules from the protozoan Oxytricha fallax share a common sequence and structure, both of which differ markedly from those deduced for yeast telomeres. Despite these differences, terminal restriction fragments from O. fallax macronuclear DNA can support telomere formation in yeasts. Two linear plasmids (LYX-1 and LYX-2) constructed by ligating BamHI-digested total Oxytricha macronuclear DNA to a yeast vector were analyzed. One end of LYX-1 and both ends of LYX-2 are derived from the Oxytricha DNA that encodes rRNA (rDNA) whereas the other end of LYX-1 is from an Oxytricha fragment other than rDNA. After propagation in yeast, both ends of LYX-1 and LYX-2 retain the C4A4 repeat characteristic of the O. fallax terminal sequence. In addition, both ends of both plasmids acquire 300-1000 base pairs of DNA containing the sequence (C-A)n, a sequence found near the termini of yeast chromosomes. Thus, at least two different Oxytricha termini display distinctive properties in yeast cells in that linear plasmids containing them are not degraded nor are they integrated into chromosomal DNA. These Oxytricha termini may act directly as telomeres in yeast; alternatively, the Oxytricha DNA may serve as a signal that results in the elaboration of a yeast telomere on the ciliate DNA.

Transposable elements as mutator genes in evolution.

Strains of the bacterium Escherichia coli harbouring genes that increase mutation rates are known to have an evolutionary advantage in chemostat competition over otherwise isogeneic strains with lower mutation rates. This advantage is frequency-dependent, the mutator strain being favoured only above a starting ratio of approximately 5 x 10(-5), and it results from the fact that the necessary beneficial mutations cannot be generated in a mutator population below a certain size. Here we consider the possibility that the mutagenic properties of transposable elements confer an advantage in the same manner as mutator genes. A previous report has shown that the transposon Tn5 increases the fitness of E. coli in chemostats, although the reason for this effect has not been established. Our results show that the transposon Tn10 also confers an advantage in chemostats. In addition, we find that (1) this advantage, like that associated with mutator genes, is frequency-dependent, (2) whenever the Tn10 strains win, a segment of Tn10, probably its IS10 sequences, has undergone transposition to a new site, (3) the new insertions converge into a site contained within a 3.2 kilobase (kb) PvuII fragment of the genome, and (4) no transpositions are detected when the Tn10 population loses. We conclude that Tn10 confers an advantage by increasing the mutation rate of the host bacterium.

The terminal organization of macronuclear DNA in Oxytricha fallax.

The macronucleus of the hypotrichous ciliate Oxytricha fallax is transcriptionally active and contains linear achromosomal DNA molecules that function as single-gene units. The terminal organization of macronuclear DNA was analyzed by chemical sequencing and S1 mapping. The terminal sequence of total macronuclear DNA was determined for molecules labeled at the 5' or 3' ends. Results indicate that the 5' sequence C4A4C4A4C4 and the 3' sequence G4T4G4T4G4T4G4T4G4 occur at both ends of all DNA molecules in the macronucleus. The discrepancy in the length of the common terminal sequence between the 5' and 3' ends was clarified by a limited S1 digestion experiment, which indicated the existence of a 16 nucleotide long single-stranded tail at the 3' ends.

The organization of macronuclear rDNA molecules of four hypotrichous ciliated protozoans.

We have compared the structure of macronuclear DNA molecules that contain rRNA genes of four hypotricous ciliates, Stylonychia pustulata, Euplotes aediculatus, Oxytricha fallax and Oxytricha nova. The macronuclear rDNA, like all macronuclear DNA in hypotrichs, exists as achromosomal molecules of approximately single-gene size. The rDNA molecules have been cloned intact as recombinant plasmids and analyzed by restriction mapping and Southern hybridization. The sites of restriction enzymes BamHI, EcoRI, HindIII, PstI, PvuII and XhoI have similar but not identical patterns in Stylonychia and the two Oxytricha rDNAs. The restriction pattern of Euplotes rDNA is unlike those of the other three, with only one site of seventeen in the same position. Despite this divergence in nucleotide sequence, the overall structure of the rDNA molecules in the four hypotrichs is constant. The size of all the rDNA molecules is the same, 7.49 kb. Also, the positions of the regions coding for 19S and 25S rRNA are alike. The 25S coding region is at the 5' end of the DNA template strand (3' end of the RNA transcript), within 500 base pairs of the terminus of the DNA molecule. The 19S coding region is adjacent to the 25S region with less than 500 base pairs of spacer lying between the two genes. The largest non-coding sequence is at the 3' end of the DNA molecule adjoining the 19S RNA gene. The 3' non-coding regions show greater sequence divergence among the different rDNAs than do the coding regions. The similarity in size and organization of these molecules and the variability in the restriction patterns suggest that the gene structure is under tighter evolutionary constraint than is the primary nucleotide sequence.

Putative actin genes in the macronucleus of Oxytricha fallax.

Previous work has shown that the macronuclear DNA of the hypotrichous ciliate Oxytricha fallax is arranged as short achromosomal pieces, 22 to 0.5 kilobase pairs (kb) in length. Micronuclear DNA has a typical chromosomal organization. Macronuclear DNA is derived from micronuclear DNA through a process of polytene chromosome fragmentation with a resultant decrease in DNA sequence complexity. Three putative actin genes have been identified in macronuclear DNA by using a cloned yeast actin gene as a hybridization probe. A restriction fragment of the yeast gene containing both actin coding and noncoding DNA hybridizes strongly to two macronuclear DNA pieces, 1.6 and 1.4 kb in length, and weakly to a 1.2-kb piece. The entire 1.6-kb piece has been cloned in plasmid pBR322 and the resulting recombinant plasmid has been designated pOfACT(1.6). The 1.6-kb pOfACT(1.6) insert hybridizes only to those restriction fragments of the yeast actin gene containing actin coding sequences. When hybridized to macronuclear DNA under conditions that allow the yeast probe to hybridize to all three macronuclear pieces, the pOfACT(1.6) insert hybridizes only to the 1.6-kb piece. Under less stringent conditions the insert also hybridizes to the 1.4-kb piece, but it shows no hybridization to the 1.2-kb DNA. The three macronuclear pieces homologous to the yeast actin gene thus differ in sequence and are interpreted as a related family of actin genes. Each of these pieces could accommodate an actin coding sequence, which in yeast, Dictyostelium discoideum, and Drosophila melanogaster is 1.1 kb, and an additional 0.1-0.5 kb of noncoding DNA.

Isolation and mapping of the rRNA genes in the macronucleus of Oxytricha fallax.

The DNA in the macronucleus of the protozoan Oxytricha, unlike like that of typical eukaryotes, exists as short, gene-sized molecules. Within the macronucleus the rRNA genes are contained in molecules 7,380 nucleotide pairs in length. This rDNA has been substantially purified by selective denaturation of non-ribosomal DNA followed by S1 nuclease digestion. Results from restriction nuclease digestion and rRNA:DNA hybridization who that the rDNA is a linear, non-palindromic molecule which contains one gene each for the 19s and 25s rRNAs. A total of less than 600 base pairs of DNA lies between the 19s and 25s genes or at the 3' end of the 25s gene. The non-coding portion of the ribosomal DNA is almost entirely limited to an approximately 1,400 base pair region at the 5' end of the molecule.

Macronuclear DNA of the hypotrichous ciliate Oxytricha fallax.

DNA in the macronuclei of Oxytricha fallax, as in other hypotrichous ciliate protozoa, exists as small, achromosomal molecules rather than in chromosomes. We report studies on O. fallax DNA using physicochemical procedures and nucleic acid hybridization. Macronuclear DNA molecules range in size from 22 kilobase pairs (kb) to about 0.5 kb. The DNA has a buoyant density in CsCl of 1.694 g.cm(-3) and a melting temperature in 15 mM NaCl/1.5 mM sodium citrate, pH 7, at 65.4 degrees . These values correspond to 34.7% Gua + Cyt and 28.1% Gua + Cyt, respectively, and base composition determined by thin-layer chromatography of nucleotides is 32.4% Gua + Cyt. The only modified nucleotide that is detectable is N(6)-methyldeoxyadenylate (0.2%), and the amount and kind of modification cannot account for the discrepancies in nucleotide composition determination by the three methods. The genes for 25S and 19S rRNA are contained in DNA molecules 6.67 kb in length, of which at least 6.15 kb is transcribed. These rDNA molecules show no intrastrand complementarity as does rDNA in some other lower eukaryotes, and they have two asymmetric sites recognized by endonuclease EcoRI. The genes for 5S RNA are in DNA molecules 0.69 kb in length. Digestion of this DNA with restriction enzymes BamHI, BsuI, HhaI, and TaqI gives no evidence for a tandemly repeated sequence. It is likely that both 19S + 25S rRNA genes and 5S RNA genes in the Oxytricha macronucleus exist as single transcription units, and both may have "spacer" regions approximately 0.5 kb long.

Intracellular localization of mouse DNA polymerase-alpha.

Although DNA polymerase-alpha (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) probably functions in the nucleus, it is usually found predominantly in the nonnuclear fraction of disrupted cells. We have reexamined the intracellular location of this enzyme using cytochalasin-B-induced enucleation, a technique which avoids exposure of nuclei to extra-cellular conditions during cell fractionation. In conditions where viability of separated cell parts is high and recovery is quantitative, we find greater than 85% of total DNA polymerase-alpha (and DNA polymerase-beta) activity in the nucleated cell fragments (karyoplasts), from which we conclude that the location in vivo of DNA polymerase-alpha is either nuclear or perinuclear. On the other hand, thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 5.7.1.75) is found primarily in the enucleated cell fragments (cytoplasts). The enucleation procedure used in this work should be of general use for intracellular location studies.

Polytene chromosomes of Oxytricha: biochemical and morphological changes during macronuclear development in a ciliated protozoan.

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120 +/- 2 polytene chromosomes and each chromosome has an average of 81 hands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus.--During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 G/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50 x 10(6) daltons to about 2 x 10(6) daltons.

DNA of ciliated protozoa: DNA sequence diminution during macronuclear development of Oxytricha.

We have measured the reassociation kinetics of DNA from the micronucleus and from the macronucleus of the hypotrichous ciliate Oxytricha. The micronuclear DNA reassociates with at least a two-component reaction, indicating the presence of both repeated and non-repeated sequences. The kinetic complexity of micronuclear non-repeated DNA is in the range of 2 to 15 X 10(11) daltons; the haploid DNA content of the micronucleus is 4 X 10(11) daltons (0.66 pg), measured microspectrophotometrically. The DNA of the macronucleus reassociates as a single second-order reaction, with a kinetic complexity of 3.6 X 10(10) daltons. A comparison of the kinetic complexities of micronuclear and macronuclear DNAs suggest a 5 to 30 fold reduction in DNA sequence complexity during the formation of a macronucleus from a micronucleus. Macronuclear DNA is in pieces with an average molecular weight of 2.1 X 10(6) daltons. Since the kinetic complexity of macronuclear DNA is 3.6 X 10(10) daltons, the macronucleus must contain about 17,000 different kinds of DNA pieces. Each macronucleus contains 3.5 X 10(13) daltons (58 pg) of DNA, indicating that each sequence must be present about 1000 times per macronucleus or 2000 times per cell.