A Novel Transvection Phenomenon Affecting the White Gene of Drosophila Melanogaster

The zeste mutation of Drosophila melanogaster suppresses the expression of white genes in the eye. This suppression is normally dependent on there being two copies of w+ located close to each other in the genome--they may either be in cis (as in a tandem duplication of w+) or in trans, i.e. on homologous chromosomes. Duplicated w+ genes carried by a giant transposing element, TE146(Z), are suppressed by z whether they are in direct (tandem) or inverted order. The tandem form of the TE is very sensitive to a rearrangement on the homologous chromosome--many rearrangements with breakpoints "opposite" the TE's insertion site prevent the interaction between the white genes on a z background. These aberrations act as dominant suppressors of zeste that are specific to the tandemly duplicated form of TE146(Z). The inverted form of the TE146(Z) presumably pairs as a hairpin loop; this is more stable than the tandem form by the criterion that its zeste phenotype is unaffected by any of the aberrations. This effect of rearrangements has been used as the basis for a screen, gamma-ray induced aberrations with at least one breakpoint opposite the TE site were recovered by their suppression of the zeste phenotype.     

DNA Repair Dependence of Somatic Mutagenesis of Transposon-Caused WHITE Alleles in DROSOPHILA MELANOGASTER after Treatment with Alkylating Agents

DNA repair-defective alleles of the mei-9, mei-41, mus-104 and mus-101 loci of Drosophila melanogaster were introduced into stocks bearing the UZ and SZ marker sets. Males with the UZ marker set, z1 (zeste allele) and w+(TE) (genetically unstable white allele presumably caused by a transposable element), or the SZ marker set, z1 and w+R (semistable white allele caused by partial duplication of the w+ locus plus transposon insert), were exposed to EMS at the first instar. After emergence, adult males bearing red spots on lemon-yellow eyes were scored as flies with somatic reversions of w+(TE) or w+R. The relative mutabilities (relative values of reversion frequency at an equal EMS dose) of either w+(TE) or w+R in a repair-proficient strain and in mei-9, mei-41, mus-104 and mus-101 strains were 1: approximately 1.2:0.3:0.3:0.7, despite the fact that w+(TE) reverted two to three times as frequently as w+R under both the repair-proficient and repair-deficient genetic conditions. Similarly, after treatment with MMS, MNNG and ENNG, w+(TE) was somatically more mutable in the mei-9 strain and less mutable in the mei-41 and mus-104 strains than in the repair-proficient strain. From these results, we propose that mutagenic lesions produced in DNA by treatment with these chemicals are converted to mutant DNA sequences via the error-prone repair mechanisms dependent on the products of the genes mei-41+ (mei-41 and mus-104 being alleles of the same locus) and mus-101+, whereas they are eliminated by mei-9+-dependent excision repair. In contrast to the approximately linear responses of induced reversions of w+(TE) with ENNG in the repair-proficient, mei-9, and mei-41 strains, seemingly there were dosage insensitive ranges for induced reversion with MNNG in the repair-proficient and mei-41 strains, but not for reversion in the mei-9 strain; w+(TE) in the mus-104 strain was virtually nonmutable with MNNG and ENNG. These results suggest that O6-methylguanine (O6MeG) produced in DNA with MNNG, but not O6-ethylguanine produced with ENNG, is almost completely repaired in a low dose range by constitutive activity of DNA O6MeG transmethylase. From the distribution of clone sizes of spontaneous revertant spots and other data, we propose that both w+(TE) and w+R have a similar tendency to spontaneously revert more frequently at early rather than at later developmental stages probably reflecting a common property of their inserted transposons.     

The genetic analysis of a large transposing element of Drosophila melanogaster

A member of Ising's family of large transposing elements (TEs) has inserted into, or very near, the crinkled (ck, 2–50) locus. This TE (TE36) carries functional alleles of both the white and roughest loci, and causes a hypomorphic mutation of ck. The TE is visible in polytene chromosomes as a two-banded insertion between 35B9 and 35C1. These bands show homology to foldback (FB) elements by in situ hybridization. All spontaneous losses of TE36 remain mutant for ck and retain sequences homologous to FB at the site of TE's insertion. TE36 carries only one functional copy of w ⁺, by the criterion that z w, TE36/ + flies are wild-type for eye color but z w; TE36/TE36 flies are zeste. This white⁺ gene is dosage compensated since w/Y; TE36/+ males have twice as much eye pigment as w/w; TE36/ + females. A form of the TE that has four polytene chromosome bands and expresses twice as much pigment as TE36 has been recovered. However, its white genes are not suppressed by zeste.     

A preliminary genetic analysis of TE146, a very large transposing element of Drosophila melanogaster

TE146 is a transposing element (TE) consisting of six polytene chromosome bands that has inserted into the no-ocelli (noc 250) locus. This member of Ising's TE family carries two copies of the white and roughest loci. TE146 is lost from noc with a spontaneous frequency of approximately 1 in 22000 chromosomes. All spontaneous losses are accompanied by the reversion of the noc mutation associated with the TE. The TE is associated with fold-back (FB) sequences. The losses of TE146 retain fold-back homology at noc. Of 26 -ray-induced losses of TE146, 16 are gross deletions, removing loci neighboring noc and ten are not. The non-deleted -ray-induced losses are either noc ^– and rst ⁺ or noc ⁺ and rst ^–. The white⁺ genes of TE146 are dosage compensated since w/Y; TE146/+ and w/w; TE146/+ flies are sexually dimorphic for eye color. These w ⁺ genes are also suppressed by zeste since z w; TE146/+ flies have zeste-colored eyes.     

Interactions between WHITE Genes Carried by a Large Transposing Element and the ZESTE Allele in DROSOPHILA MELANOGASTER

TE146, a large transposing element of Drosophila melanogaster, carries two copies of the white and roughest genes in tandem. In consequence, z¹ w ^11E4; TE146(Z)/+ flies have a zeste (lemon-yellow) eye color. However, one in 10³ TE146 chromosomes mutates to a red-eyed form. The majority of these "spontaneous red" (SR) derivatives of TE146 have only one copy of the white gene and are, cytologically, two- to three-banded elements, rather than six-banded as their progenitor. The SR forms of TE146 are also unstable and give zeste-colored forms with a frequency of about one in 10⁴. One such "spontaneous zeste" (SZ) derivative carries duplicated white genes as an inverted, rather than a tandem, repeat. The genetic instability of this inverted repeat form of TE146 is different from that of the original tandem repeat form. In particular, the inverted repeat form frequently produces derivatives with internal rearrangements of the TE and gives a much lower frequency of SR forms. In addition, two novel features of the interaction between w⁺ alleles in a zeste background have been found. First, copies of w ⁺ can become insensitive to suppression by zeste even when paired. Second, an inversion breakpoint may disrupt the pairing between two adjacent w⁺ alleles, necessary for their suppression by zeste, without physically separating them.     

Isolation of yeast histone genes H2A and H2B

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.     

Unusual suppression properties of lysogens containing derivatives of ?80 psu3+

Summary Nonsense codon suppressing lysogens of E. coli have been made using 80 psu ₃ ⁺ -A2 and 80 psu _oc ⁺ -A2, heat-sensitive amber and ochre suppressing derivatives, respectively, of bacteriophage 80. The various lysogens selected differ in strength of suppression as well as in heat sensitivity of suppressor function. Heat-resistant derivatives, some still carrying the A2 mutation, can be selected from the heat sensitive parents. Mapping expreiments indicate that the 80 derivatives integrate at the tyrTV locus, which contains two copies of tRNA ₁ ^Tyr . The origin of the various suppressor phenotypes appears to be related to the great variety of distinctive recombination events possible either between the incoming tRNA ₁ ^Tyr gene and the host copies, or among the three copies of this gene in the lysogens.     

Mapping of a Bradyrhizobium japonicum DNA Region Carrying Genes for Symbiosis and an Asymmetric Accumulation of Reiterated Sequences

Spontaneous kanamycin-sensitive derivatives were obtained from Bradyrhizobium japonicum (strain 110) carrying Tn5 insertions in symbiotic gene cluster I; the derivatives were shown to have deletions of cluster I plus flanking DNA which was indicated by the absence of different copies of the repeated sequences RSα and RSβ. The deletion endpoints were mapped using cloned wild-type DNA fragments containing RSα copies which also served as origins for overlapped cosmid cloning. The majority of the deletions resulted from recombinational fusion of two remote RSα copies. Novel types of repeated sequences (RSγ, RSδ, and RSε) occurring in 12, 10, and 4 copies per genome were detected. Seven, nine, and three copies of RSγ, RSδ, and RSε, respectively, were located near cluster I. It is concluded that the B. japonicum genome has an unusual DNA segment of >230 kilobase pairs characterized by the presence of repeated sequences and genes for symbiotic N₂ fixation.     

A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination.

We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance). The procedure employs the high level of transformability of B. subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B. subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus. In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected. Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene. Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.     

Isolation and characteristics of a temperature-sensitive plasmid pRP19.6--an RP1 derivative containing the duplicated IS21 sequence

When analysing the antibiotic resistant, temperature-independent derivatives of Proteus mirabilis cells, carrying the plasmid RP1ts12, a derivative of the latter (pRP19.6) with an elevated frequency of integration into E. coli K12 chromosome, has been isolated. The structure and properties of pRP19.6 was studied. As revealed from the data of structural and genetic analyses pRP19.6 is identical to the factor R68.45 described earlier by Haas and Holloway. Similarly to R68.45, the plasmid under study contains two copies of IS21 sequence and mobilises nonconjugative plasmid pBR325 with high efficiency. Using the temperature sensitive replication of pRP19.6, frequency of it's integration into the chromosomes of E. coli rec+ and recA- stains is determined. It is demonstrated that the clones carrying the plasmid in integrated state are Hfr-strains. The possibilities to use the temperature sensitive R68.45 like plasmid for isolation of Hfr-strains in the broad range of gram-negative bacteria and for insertional inactivation of chromosomal genes are discussed.     

Genetic mapping of Vibrio cholerae enterotoxin structural genes

The structural genes which constitute the cholera toxin operon, ctxAB, were genetically mapped in the Vibrio cholerae El Tor strain RV79. This strain of V. cholerae contains two copies of the ctx operon located on a 7-kilobase-pair tandemly duplicated region. We began by isolating a vibriophage VcA1 insertion mutation in one of the two ctxA genes located in this region. The mutant carrying this ctxA::VcA1 insertion, DC24, was converted to a VcA1-facilitated donor by introduction of the conjugal plasmid pSJ15, which carries an inserted copy of a defective VcA1-like prophage. The donor characteristics of DC24(pSJ15) indicated that the ctxA::VcA1 insertion mutation was near the trp region of the V. cholerae chromosome. Subsequent RV79 three-factor crosses were performed between VcA1-facilitated donors and recipient strains carrying one of two structural gene mutations in ctx, either delta ctxA23P Kmr or delta ctx-7922. The former was constructed by an in vivo marker exchange procedure and could be scored either by its kanamycin resistance phenotype or by its lack of DNA sequences homologous to the ctxA region. The delta ctx-7922 mutation is a total deletion of both ctx copies of strain RV79. The three-factor cross data strongly suggest that the two ctx loci of RV79 map between the nal and his genes of V. cholerae in the trp nal his linkage group. Physical analysis and heterologous crosses between an RV79 El Tor donor and a 569B classical recipient indicates that one of the two 569B ctx operon copies maps in the same region as the RV79 ctx loci (i.e., linked to nal). Together with previously published observations, these data show that the ctx structural genes are not closely linked to other genes known to affect toxin production in V. cholerae.     

Fitness costs of Doc expression are insufficient to stabilize its copy number in Drosophila melanogaster

The stable coexistence of transposable elements (TEs) with their host genome over long periods of time suggests TEs have to impose some deleterious effect upon their host fitness. Three mechanisms have been proposed to account for the deleterious effect caused by TEs: host gene interruptions by TE insertions, chromosomal rearrangements by TE-induced ectopic recombination, and costly TE expression. However, the relative importance of these mechanisms remains controversial. Here, we test specifically if TE expression accounts for the host fitness cost imposed by TE insertions. In the retrotransposon Doc, expression requires binding of the host RNA polymerase to the internal promoter. If expression of Doc elements is deleterious to their host, Doc copies with promoters would be more strongly selected against and would persist in the population for shorter periods of time compared with Docs lacking promoters. We tested this prediction using sequence-specific amplified polymorphism (SSAP) analyses. We compared the populations of these two types of Doc elements in two sets of lines of Drosophila melanogaster: selection-free isogenic lines accumulating new Doc insertions and isogenized isofemale lines sampled from a natural population. We found that (1) there is no difference in the proportion of promoter-bearing and promoter-lacking copies between sets of lines, and (2) the site occupancy distribution of promoter-bearing copies does not skew toward lower frequency compared with that of promoter-lacking copies. Thus, selection against promoter-bearing copies does not appear to be stronger than that of promoter-lacking copies. Our results show that expression is not playing a major role in stabilizing Doc copy numbers.     

Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes.

We cloned and sequenced the second gene coding for yeast ribosomal protein 51 (RP51B). When the DNA sequence of this gene was compared with the DNA sequence of RP51A (J.L. Teem and M. Rosbash, Proc. Natl. Acad. Sci. U.S.A. 80:4403--4407, 1983), the following conclusions emerged: both genes code for a protein of 135 amino acids; both open reading frames are interrupted by a single intron which occurs directly after the initiating methionine; the open reading frames are 96% homologous and code for the same protein with the exception of the carboxy-terminal amino acid; DNA sequence homology outside of the coding region is extremely limited. The cloned genes, in combination with the one-step gene disruption techniques of Rothstein (R. J. Rothstein, Methods Enzymol. 101:202-211, 1983), were used to generate haploid strains containing mutations in the RP51A or RP51B genes or in both. Strains missing a normal RP51A gene grew poorly (180-min generation time versus 130 min for the wild type), whereas strains carrying a mutant RP51B were relatively normal. Strains carrying mutations in the two genes grew extremely poorly (6 to 9 h), which led us to conclude that RP51A and RP51B were both expressed. The results of Northern blot and primer extension experiments indicate that strains with a wild-type copy of the RP51B gene and a mutant (or deleted) RP51A gene grow slowly because of an insufficient amount of RP51 mRNA. The growth defect was completely rescued with additional copies of RP51B. The data suggest that RP51A contributes more RP51 mRNA (and more RP51 protein) than does RP51B and that intergenic dosage compensation, sufficient to rescue the growth defect of strains missing a wild-type RP51A gene, does not take place.     

US specificity of occasion setting: Hierarchical or configural learning?

Four experiments in rats examined whether occasion setters and target CSs play qualitatively different roles in occasion-setting discriminations. Two visual occasion setters, A and B, signalled reinforcement of two auditory target CSs, x and y, with sucrose and oil (A…x→suc, B…y→oil, A-, B-, x-, y-); in addition two transfer CSs w and z were paired with sucrose and oil (w→suc, z→oil). When w and z were substituted for x and y (A…w, B…w, A…z, B…z) more responding was observed when both stimuli had been paired with the same outcome (Experiments 1 and 3a). No effect was observed when two visual "pseudo-occasion setters", C and D (paired with sucrose and oil in a trace relation to the US:C…→suc, D…→oil), were substituted for the occasion setters A and B (C…x, D…x, C…y, D…y; Experiments 2, 3b and 4). These results could not be explained in terms of Pavlovian summation: responding to combinations of Pavlovian CSs paired with same or different outcomes was either the same, or lower when both stimuli had been paired with the same outcome (Experiment 4). Implications of these results for theories of occasion setting and configural learning are discussed.     

When is evolutionary branching in predator-prey systems possible with an explicit carrying capacity?

In this study we use the theory of adaptive dynamics firstly to explore the differences in evolutionary behaviour of a generalist predator (or more specifically an omnivorous or intraguild predator) in a predator-prey model, with a Holling Type II functional response, when two distinct forms for the carrying capacity are used. The first of these involves the carrying capacity as an emergent property, whilst in the second it appears explicitly in the dynamics. The resultant effect this has on the intraspecific competition in each case is compared. Taking an identical trade-off in each case, we find that only with an emergent carrying capacity is evolutionary branching possible. Our study then concentrates solely on the case where the carrying capacity appears explicitly. Using the same model as above, but choosing alternate trade-offs, we find branching can occur with an explicit carrying capacity. Our investigation finishes by taking a more general functional response in an attempt to derive a condition for when branching can or cannot occur. For a predator-prey model, branching cannot occur if the functional response can be separated into two components, one a function of the population densities, X and Z, and the other a function of the evolving parameter z (traded off against the intrinsic growth rate), i.e. if F(z,X,Z) = F(1)(z)F(2)(X,Z). This search for evolutionary branching is motivated by its possible role in speciation.     

The Molecular Structure of Te146 and Its Derivatives in Drosophila Melanogaster

TE146 is a giant transposon of Drosophila melanogaster. It carries two copies of the white and roughest genes, normally found on the X chromosome. The structure of this transposon has been studied at the molecular level. TE146 may transpose to new chromosome positions, excise and be lost from the genome or undergo internal rearrangements. The termini of TE146 are foldback DNA elements (FB); the transposon also carries two internal FB elements. Loss or internal rearrangement of TE146 involves recombination between different FB elements. These events have been mapped molecularly, by taking advantage of the fact that the FB sequences are composed largely of a regular 155-bp repeat sequence that is cut by the restriction enzyme TaqI, and are shown to be nonrandom. We suggest that these FB-FB exchange events occur by mitotic sister-chromatid exchange in the premeiotic germ line.