Lethality Patterns and Morphology of Selected Lethal and Semi-Lethal Mutations in the Zeste-White Region of DROSOPHILA MELANOGASTER

Aspects of the developmental genetics of lethal and semi-lethal mutants representing 13 complementation groups (cistrons) in the 3A-3C region of the X chromosome of Drosophila melanogaster are given. Each of these cistrons is associated with a particular chromomere in the salivary gland chromosome. Mutants within each cistron have similar lethality patterns and morphological attributes, and the characteristics of a given cistron are distinct with respect to other cistrons. These results provide additional evidence that only one function is associated with each chromomere.-The results of the lethality pattern analysis are also compared with previous studies of lethal mutants of Drosophila.     

Origin and direction phiX174 double- and single-stranded DNA synthesis

The origin and direction of both φX174 double-stranded and single-stranded DNA synthesis has been determined by pulsing replicating viral DNA molecules with [³H]thymidine for periods of less than one round of DNA synthesis and examining distribution of activity in the Haemophilus influenzae restriction endonuclease (Hin) DNA fragments of these molecules. In early RFI and RFII DNA intermediates in double-stranded DNA replication, gradients of label were observed which started in the R3 fragment (cistron A) and increased towards the R4 fragment (cistron H). The origin of synthesis is near the R4/R3 junction of the R3 fragment. Thus, φX174 double-stranded DNA synthesis proceeds clockwise around the genetic map (5′ → 3′), in one direction only and starting in the region of cistron A, a conclusion consistent with the genetic experiments of Baas &; Jansz (1972). Similar experiments with the gapped late RFII DNA molecules that have just completed a round of single-stranded viral DNA synthesis demonstrated that φX174 single-stranded DNA synthesis also has a single origin of replication in the region of cistron A, and that the synthesis moves in the 5′ → 3′ direction, around the genetic map. The gap in both the early and the late RFII DNA molecules also appears to be in the R3 fragment containing cistron A.     

Functional and structural units in the chromomere

Electron microscopic observations demonstrate the existence of several DNA packing levels in the chromomere. A linear DNA molecule forms a big (chromomere) loop anchored to the chromosomal scaffold. The loop forms a set of smaller loops in the rosette pattern. Packing of the DNA by the histone octamer particles results in nucleosomes and nucleomeres. To establish the possible correspondence between the structural units of a chromomere and the genetical units (genes, exons, introns) in it, we compared the lengths of the units. Statistical analysis of the 315 sequenced genes indicate that the average gene size corresponds to the average length of a rosette loop. It means that a chromomere contains one or more genes. Assuming that exon-intron boundaries cannot bind nucleosomes we constructed DNA-packing models of the 88 genes. They demonstrate that the first (in 77.8 per cent of the genes) and the last (in 52.7 per cent) exons of the genes are too short to bind nucleosomes. Many genes contain long (nucleosome binding) pieces of DNA. Long packed pieces are introns in vertebrates; they are exons in invertebrates and plants. The average size gene contains two nucleomeres.     

Genetic manipulation of cyclic AMP levels in Drosophila melanogaster.

Cyclic AMP levels in $\text{Drosophila}$,$\text{melanogaster}$ adults can be altered genetically by changing the number of doses of chromomere 3D4 contained in the genome, a chromomere previously shown to control the activity of cyclic AMP phosphodiesterase in a dose-dependent manner. Flies completely deficient for chromomere 3D4 have 2–7 times the cyclic AMP level of flies with one or two doses of chromomere 3D4. Cyclic AMP levels are significantly depressed in flies carrying three doses of 3D4. Cyclic GMP levels are not influenced in a dose-dependent manner by chromomere 3D4. The effect on cyclic AMP levels may provide a useful system for investigating physiological and developmental consequences of aberrant cyclic AMP levels in the intact organism.     

Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1

Transfer-defective mutants of the 10.4-kb Tra 2/Tra 3 region of RP1 were identified by their ability to be complemented by clones carrying all or part of this region. The respective mutations occurred in six cistrons whose order (traA, B, E, R, P, Q) and location were determined by deletion and insertion mapping. The cistrons occupy a minimum of 5.5 kb with the most distal, traA, spanning the 28.0-kb map position and traR the KpnI site at map position 24.1 kb. Each cistron is expressed independently, as Tn5 or Tn504 insertions in any one cistron do not affect the other five. The phenotypes controlled by each cistron suggest that all contribute to pilus biosynthesis/function while three (traB, R, and P) also contribute to surface exclusion. Given the occurrence of tra cistrons in the "silent" region between Tra 2 and Tra 3 we propose that the epithet "Tra 2" should be used to describe this entire region.     

Effects of Uvsx, Uvsy and DNA Topoisomerase on the Formation of Tandem Duplications of the Rii Gene in Bacteriophage T4

We have characterized tandem duplications in the rII regions of phage T4. The rII deletion r1589 blocks only the function of the rIIA cistron, although it extends into the B cistron. Another rII deletion, r1236, blocks the function of the rIIB cistron and overlaps r1589. When a cross is made between r1589 and r1236, true rII(+) progeny cannot form. Instead, anomalous phenotypically rII(+) phages are detected carrying an rII region from each parent. Analyses of nucleotide sequences of the recombination junctions indicate that recombination takes place between short regions of homology (from 2 to 10 bp). Open reading frames of the recombinants deduced from the nucleotide sequences reveal that they contain a normal rIIA cistron and one of a variety of fused, duplicated rIIB cistrons. The T4 uvsX and uvsY genes, which participate in homologous recombination, are involved in this duplication formation. T4 DNA topoisomerase is encoded by genes 39, 52 and 60. Mutations in 52 and 60 reduced the frequency of such duplications, but mutations in gene 39 and some in gene 52 did not. Hence, the effects of topoisomerase mutations are allele-specific. Models are proposed in which these proteins are involved in tandem duplication.     

Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora erythrocephala

We have examined the possibility that the intron-containing (intron⁺) rDNA cistrons of Dipteran flies are active in the germ-line derived polyploid nurse cell nuclei of the ovarian follicles. Using the organism, Calliphora erythrocephala, we describe here a procedure which yields very pure nurse cell nuclei and compare the intron-free (intron^–) and intron⁺ rDNA cistron contents of nurse cell nuclei prepared by this procedure to those of 2–18 h embryo nuclei and 3 day pupal nuclei. DNA from three preparations of each nuclear type was examined and the intron^– and intron⁺ cistron contents quantitated using a Southern transfer procedure. The number of intron^– and intron⁺ rDNA cistrons per haploid genome in the presumed diploid 2–18 h embryo DNA was first established, and then the intron^– and intron⁺ cistron contents of nurse cell nuclear DNA and 3 day pupal DNA were determined relative to these values.The intron^– cistron content of nurse cell nuclear DNA was indistinguishable from that of embryonic DNA but the intron⁺ cistrons showed an 8-fold under-replication relative to the presumed diploid DNA. A slight under-representation of the intron^– cistrons and 3-fold under-replication of the intron⁺ cistrons were demonstrated for 3 day pupal DNA. These findings strongly suggest that intron⁺ rDNA cistrons are non-functional in nurse cell nuclei and substantiate the generality of this implication for the whole organism during early pupal life.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. III. Physical location of mitochondrial genetic markers and the molecular nature of omega.

1. We have determined the physical location of mitochondrial genetic markers in the 21S region of yeast mtDNA by genetic analysis of petite mutants whose mtDNA has been physically mapped on the wild-type mtDNA. 2. The order of loci, determined in this study, is in agreement with the order deduced from recombination analysis and coretention analysis except for the position of omega+: we conclude that omega+ is located between C321 (RIB-1) and E514 (RIB-3). 3. The marker E514 (RIB-3) has been localized on a DNA segment of 3800 bp, and the markers E354, E553 and cs23 (RIB-2) on a DNA segment of 1100 base pairs; both these segments overlap the 21S rRNA cistron. The marker C321 (RIB-1) has been localized within a segment of 240 bp which also overlaps the 21S rRNA cistron, and we infer on the basis of indirect evidence that this marker lies within this cistron. 4. In all our rho+ as well as rho- strains there is a one-to-one correlation between the omega+ phenotype, the ability to transmit the omega+ allele and the presence of a mtDNA segment of about 1000 bp long, located between sequences specifying RIB-3 and sequences corresponding to the loci RIB-1 and RIB-2. This segment may be inserted at this same position into omega- mtDNA by recombination. 5. The role which the different allelic forms of omega may play in the polarity of recombination is discussed.     

Structure of an intermediate in the replication of bacteriophage φX174 deoxyribonucleic acid: The initiation site for DNA replication

Replicative form DNA composed of a closed complementary strand and a discontinuous viral strand has been isolated from cells infected with bacteriophage φX174 during the period of single-strand DNA synthesis. This RFII DNA was degraded by the restriction enzyme from Hemophilus influenzae, endonuclease R, and the products analyzed by polyacrylamide gel electrophoresis. The results indicate that there are two types of discontinuity in the viral strands of these molecules: (1) 65% of the molecules contain a gap, which causes a discrete increase in mobility of a specific restriction enzyme fragment, R3. This gap can be selectively repaired with Escherichia coli DNA polymerase I and nucleoside triphosphates, but the molecules are not converted to RFI by addition of E. coli polynueleotide ligase to the reaction mixture. Approximately 30 moles of radioactive TTP are incorporated per mole of RF DNA. (2) 35% of the RF molecules contain a discontinuity, which does not result in a detectable change in mobility of any restriction enzyme fragment. These RF molecules can be converted to RFI by the action of ligase and polymerase I in the presence of nucleoside triphosphates, with incorporation of only approximately one mole of radioactive TTP, specifically into fragment R3, per mole of RF DNA.When the reaction of late RFII DNA and polymerase I is allowed to proceed beyond the repair of the discontinuity, radioactive nucleotides are incorporated into endonuclease R fragments adjacent to R3 in the 5′ → 3′ direction. This technique was utilized to determine a partial order of endonuclease R fragments in φX174.These results suggest that the synthesis of single-strand DNA is initiated from a unique point in cistron A and proceeds clockwise round the φX174 genetic map (cistron order: ABCDEFGH). A comparison of these results with other studies on φX174 suggests that DNA synthesis in all stages of φX174 replication may be initiated from a specific locus on the genome, at or near cistron A.     

LINKAGE OF 5S RIBOSOMAL DNA TO OTHER rDNAS IN THE CHROMOPHYTIC ALGAE AND RELATED TAXA1

Gene organization within the nuclear ribosomal DNA cistron and linkage of the 5S rDNA gene to the cistron were surveyed in 20 taxa of protists representing most of the Chromophyta (stramenopiles) and representatives of the Dinophyceae (alveolata) and Euglenophyceae. The intergenic spacer, which separates adjacent cistrons, was first PCR-amplified from total DNA using primers anchored in the 3’end of the large subunit and the 5’end of the small subunit in the next downstream cistron. Presence of the 5S gene in the cistron was determined by a second round of PCR using primers anchored in the large subunit and the 5S gene. where 5S-linked rDNA was not detected in the cistron, the presence of 5S tandem repeating units were confirmed by the PCR of 5S-5S fragments from the total DNA. Results show that most of the Chromophyta, as well as Opalina, Proteromonas (colorless stramenopiles), Dinophyceae, and Euglenophyceae have a 5S-linked type of rDNA organization. In contrast, only tandem repeats of 5S rDNA were detected in Bacillariophyceae and Synurophyceae. The occurrence of 5S-unlinked rDNA is hypothesized to be the result of secondary transfer from an ancestral, linked 5S type. The 5S-linked type of rDNA organization is apparently common in protists. Given the fact that most of these protists have mitochondria with tubular or discoid cristae, as compared with flattened cristae common in higher plants and animals, we conclude that the 5S-linked type of rDNA diverged at a very early stage in the evolution of eukaryotes.     

Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function.

The main binding site for mammalian ribosomes on the single-stranded RNA of bacteriophage MS2 is located nine tenths of the way through the coat protein gene. Translation initiated at an AUG triplet in the +1 frame yields a 75 amino acid polypeptide which terminates within the synthetase gene at a UAA codon, also in the +1 frame. Partial amino acid sequence analysis of the product synthesized in relatively large amounts by mammalian ribosomes confirms this assignment of the overlapping cistron. The same protein is made in an E. coli cell-free system, but only in very small amounts. Analysis of the translation products directed by RNA from op3, a UGA nonsense mutant of phage f2, identifies the overlapping cistron as a lysis gene. In this paper we show that the op3 mutation is a C yield U transition occurring in the second codon of the synthetase cistron, which explains the lowered production of phage replicase (as well as lack of lysis) upon op3 infection of nonpermissive cells. We discuss the properties of the overlapping gene in relation to its lysis function, recognition of the lysis initiator region by E. coli versus eucaryotic ribosomes and op3 as a ribosome binding site mutant for the f2 synthetase cistron.     

Conservation of the sequence and position of the ribosomal RNA genes in Tetrahymena pyriformis mitochondrial DNA.

1. We have done cross-hybridizations between the mitochondrial ribosomal RNAs and DNAs from strains ST and PP of Tetrahymena pyriformis. DNA . ribosomal RNA hybrid formation can be completely prevented by an excess of the heterologous ribosomal RNA and the heterologous hybrids melt 6 degrees C below the homologous hybrids. This shows that the ribosomal RNA cistrons can account for the 5% cross-hybridization previously observed between the mtDNAs of strains PP and ST (Goldbach et al. (1977) Biochim. Biophys. Acta 477, 37--50). 2. By electron microscopy of DNA . ribosomal RNA hybrids we have determined the position of the ribosomal RNA cistrons on the mtDNA of strain GL, a mtDNA which we have shown to contain a sub-terminal 1 micron duplication-inversion and a terminal palindrome at one end which varies in length from 0 to 5 micron and which includes the 1 micron duplication-inversion (Arnberg et al. (1977) Biochim. Biophys. Acta 477, 51--69). The 21 S ribosomal RNA cistron overlaps the 1 micron duplication-inversion and as a result two or three cistrons are present, depending on the size of the terminal palindrome. Only one 14 S ribosomal RNA cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron and with the same polarity as this cistron. 3. We conclude from these results and those in the preceding paper that the sequence of the ribosomal RNAs and the position of the ribosomal RNA genes in the mtDNA is strongly conserved in Tetrahymena. Possible reasons for the duplication of 21-S ribosomal RNA genes and the terminal heterogeneity of Tetrahymena mtDNA are discussed.     

High intra-individual sequence variation in the nuclear rDNA LSU-5S intergenic spacer in the Sargassaceae (Fucales, Phaeophyceae)

Of four species of Sargassaceae, representing the genera Carpophyllum, Cystoseira, Landsburgia, and Sargassum, the intergenic spacer of the ribosomal cistron was amplified using a forward primer annealing at the 3′-end of the large subunit (LSU) of the ribosomal cistron and a reverse primer annealing at the 5S rRNA gene. The PCR products were cloned and the DNA sequences of multiple clones were determined. Almost each clone showed a unique DNA sequence. Intra-individual variation of this LSU-5S intergenic spacer was extremely high and was characterized by great length variation and a high number of short tandem repeats. Sequences were unalignable and therefore it was concluded that the LSU-5S intergenic spacer is unsuitable for phylogenetic and phylogeographic studies of sargassacean taxa.     

φX-174 Bacteriophage Structural Mutants Which Affect Deoxyribonucleic Acid Synthesis

Seven cistrons in X-174 were identified and one in particular was studied intensively: cistron A, which is assigned a protein in the mature phage. Amber mutants in this cistron synthesize a new deoxyribonucleic acid (DNA) form in addition to circular phage DNA upon infection of the restrictive host. This DNA is linear, non-infectious, and single-stranded; it is formed from the phage strand of replicative form X-174 DNA. These mutants produce two different defective particles in the restrictive host. One particle contains circular phage DNA but is not infectious; the other contains the new DNA form and is similar to the 70S particles found in wild-type phage lysates. The mutant A gene product acts independently of normal A protein upon mixed infection of the restrictive host with an A mutant and a mutant from any other cistron or wild type.     

The Product of the LEU-3 Cistron as a Regulatory Element for the Production of the Leucine Biosynthetic Enzymes of Neurospora

A class of intracistronic (or closely linked) partial reversions of leu-3 mutations has been found to be conditionally constitutive with respect to the synthesis of isopropylmalate isomerase (specified by the leu-2 cistron) and beta-isopropylmalate dehydrogenase (specified by the leu-1 cistron), two of the enzymes of leucine biosynthesis in Neurospora. The intermediate level of enzyme production by these leu-3(cc) mutants is independent of the obligatory inducer effector, alpha-isopropylmalate, but dependent upon the presence of the branched-chain amino acids, isoleucine, valine and leucine. The properties of leu-3+, leu-3 and leu-3(cc) in heterokaryons indicate that the transnuclear regulatory activity of the leu-3 product varies specifically as a function of available effector molecules. The information presented suggests that the leu-3 cistron is responsible not only for the production of a "positive" regulatory substance necessary for the expression of the leu-1 and leu-2 cistrons, but that it probably serves also a coordinating role in the expression of many of the genes involved in branched-chain amino acid metabolism.