Heterochromatin protein 1 binds transgene arrays.

Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation.

Heterochromatic trans-inactivation of Drosophila white transgenes.

Position effect variegation of most Drosophila melanogaster genes, including the white eye pigment gene is recessive. We find that this is not always the case for white transgenes. Three examples are described in which a lesion causing variegation is capable of silencing the white transgene on the paired homologue (trans-inactivation). These examples include two different transgene constructs inserted at three distinct genomic locations. The lesions that cause variegation of white minimally disrupt the linear order of genes on the chromosomes, permitting close homologous pairing. At one of these sites, trans-inactivation has also been extended to include a vital gene in the vicinity of the white transgene insertion. These findings suggest that many Drosophila genes, in many positions in the genome, can sense the heterochromatic state of a paired homologue.

Transgene repeat arrays interact with distant heterochromatin and cause silencing in cis and trans.

Tandem repeats of Drosophila transgenes can cause heterochromatic variegation for transgene expression in a copy-number and orientation-dependent manner. Here, we demonstrate different ways in which these transgene repeat arrays interact with other sequences at a distance, displaying properties identical to those of a naturally occurring block of interstitial heterochromatin. Arrays consisting of tandemly repeated white transgenes are strongly affected by proximity to constitutive heterochromatin. Moving an array closer to heterochromatin enhanced variegation, and enhancement was reverted by recombination of the array onto a normal sequence chromosome. Rearrangements that lack the array enhanced variegation of white on a homologue bearing the array. Therefore, silencing of white genes within a repeat array depends on its distance from heterochromatin of the same chromosome or of its paired homologue. In addition, white transgene arrays cause variegation of a nearby gene in cis, a hallmark of classical position-effect variegation. Such spreading of heterochromatic silencing correlates with array size. Finally, white transgene arrays cause pairing-dependent silencing of a non-variegating white insertion at the homologous position.

Gene family use and somatic mutation in primary and secondary fluorescein-specific IgM antibody responses.

A comparative analysis of the DNA sequences of primary and secondary IgM, fluorescein-specific antibodies was performed. These antibodies were secreted by hybridomas generated following fusion of immunized BALB/c mouse lymphocytes and SP2/0 myeloma cells. Our results show that primary and secondary fluorescein-specific IgM antibodies use a variety of segments from the variable region of the immunoglobulin heavy chain locus (VH), with members of the J558 and 7183 VH gene families predominating in both populations. D regions from the DF116 and DSP2 families were used exclusively in our primary antibody sample and predominated in the secondary response. In the primary antibodies, 15 out of 18 definable D regions were transcribed in reading frame one, but in the secondary antibodies the three reading frames were used stochastically. Secondary IgM antibodies showed a higher frequency of somatic mutation than their primary counterparts, but we could detect no evidence of selection for mutations in the complementarity determining regions as compared with the framework regions. It appears that fusion of secondary cells, 3-6 days after immunization, is able to 'capture' the IgM-producing population of B cells at a stage in their development following mutation but prior to antigenic selection.

The Triplo-lethal locus of Drosophila: reexamination of mutants and discovery of a second-site suppressor.

In the genome of Drosophila melanogaster there is a single locus, Triplo-lethal (Tpl), that causes lethality when present in either one or three copies in an otherwise diploid animal. Previous attempts to mutagenize Tpl produced alleles that were viable over a chromosome bearing a duplication of Tpl, but were not lethal in combination with a wild-type chromosome, as deficiencies for Tpl are. These mutations were interpreted as hypomorphic alleles of Tpl. In this work, we show that these alleles are not mutations at Tpl; rather, they are dominant mutations in a tightly linked, but cytologically distant, locus that we have named Suppressor-of-Tpl (Sul(Tpl)). Su(Tpl) mutations suppress the lethality associated with three copies of the Triplo-lethal locus and are recessive lethal. We have mapped Su(Tpl) to the approximate map position 3-46.5, within the cytological region 76B-76D.

Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila.

Closely linked repeats of a Drosophila P transposon carrying a white transgene were found to cause white variegation. Arrays of three or more transgenes produced phenotypes similar to classical heterochromatin-induced position-effect variegation (PEV), and these phenotypes were modified by known modifiers of PEV. This effect on the repeated transgenes was much stronger for a site near centric heterochromatin than it was for a medial site, and it strengthened with increasing copy number. Differences between variegated phenotypes could be accounted for if different topological structures were generated by pairing between closely linked repeat sequences. We propose that pairing of repeats underlies heterochromatin formation and is responsible for diverse gene silencing phenomena in animals and plants.

Suppression of a lethal trisomic phenotype in Drosophila melanogaster by increased dosage of an unlinked locus.

One of the most extreme examples of gene dosage sensitivity is the Triplo-lethal locus (Tpl) on the third chromosome of Drosophila melanogaster, which is lethal when present in either one or three copies. Increased dosage of an unlinked locus, Isis, suppresses the triplo-lethal phenotype of Tpl, but not the haplo-lethal phenotype. We have mapped Isis to the X chromosome region 7E3-8A5, and shown that the suppression is a gene dosage effect. Altered dosage of Isis in the presence of two copies of Tpl has no obvious effects. By examining the interactions between Isis dosage and Tpl we suggest that Isis does not directly repress Tpl expression, but acts downstream on the triplo-lethal phenotype of Tpl.

Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster.

A locus associated with a severe Minute effect has been mapped at 7C on the X chromosome of Drosophila melanogaster. Previous work has suggested that this Minute encodes ribosomal proteins S14A and S14B. We have made a chromosomal deficiency that removes the S14 ribosomal protein genes, yet does not display the Minute phenotype. These data suggest that the S14 genes do not actually correspond to the Minute locus.

Temporal variations in the fine specificity of IgM anti-fluorescyl antibodies.

This study compares the fine specificities of the primary and secondary fluorescein (FITC)-specific immunoglobulin M (IgM) repertoires in BALB/c mouse serum and monoclonal antibodies (MoAb) and has found reproducible, immunization-dependent differences. FITC and four of its homologues; iodoacetamido fluorescein (IAF), dichlorotriazinyl aminofluorescein (DTAF), substituted rhodamine isothiocyanate (XRITC) and tetramethyl rhodamine isothiocyanate (TRITC), each conjugated to bovine serum albumin (BSA), were used to determine reactivity patterns of serum IgM from mice immunized once or twice with FITC-haemocyanin (FITC-Hy). Reactivity patterns were also obtained for 20 IgM MoAb, eight of which were produced by fusions of SP2/0 myeloma cells with splenocytes from mice immunized once (primary) and 12 from mice immunized twice (secondary) with FITC-Hy. Each MoAb exhibited a unique fine specificity pattern, evidence of extensive heterogeneity in the FITC-specific repertoire. Reactivities of IgM MoAb with certain homologues were found to be more characteristic of either the primary or secondary response. Polyclonal serum IgM also showed reproducible immunization-dependent variations in fine specificity. Such a pattern could result from idiotypic suppression of primary antibodies, from the expansion of subsets of IgM memory cells utilizing novel genes and/or from somatic mutation absent in primary IgM antibodies.

A novel RNA helicase gene tightly linked to the Triplo-lethal locus of Drosophila.

The Triplo-lethal (Tpl) locus of Drosophila is the only known locus which is lethal when present in three copies rather than the normal two. After recovering a hybrid-dysgenesis-induced mutation of Tpl we used a rapid combination of transposon tagging, chromosome microdissection and PCR to clone the P element that had transposed into the Tpl region. That P element is located within the gene for a new and unique member of the RNA helicase family. This new helicase differs from all others known by having glycine-rich repeats at both the amino and carboxyl termini. Curiously, genetic analysis shows that the P element inserted into this gene is not responsible for the Tpl mutant phenotype. We present possible explanations for these findings.

The unusual spectrum of mutations induced by hybrid dysgenesis at the Triplo-lethal locus of Drosophila melanogaster.

The Triplo-lethal locus (Tpl) is unique in its dosage sensitivity; no other locus in Drosophila has been identified that is lethal when present in three doses. Tpl is also haplo-lethal, and its function is still a mystery. Previous workers have found it nearly impossible to mutationally inactive Tpl other than by completely deleting the chromosomal region in which Tpl resides (83DE). We have utilized P-M hybrid dysgenesis in an effort to obtain new mutations of Tpl. We recovered 19 new duplications of Tpl, 15 hypomorphic mutations of Tpl (a previously rare class of mutation), and no null mutations. Surprisingly, 14 of the 15 hypomorphic alleles have no detectable P element sequences at the locus. The difficulty in recovering null mutations in Tpl suggests that it may be a complex locus, perhaps consisting of several genes with redundant functions. The relative ease with which we recovered hypomorphic alleles is in sharp contrast to previous attempts by others to mutagenize Tpl. A higher mutation rate with hybrid dysgenesis than with radiation or chemicals also suggests a peculiar genetic organization for the locus.

Antigenic requirements for stimulation and target cell recognition by immune and non-immune cytotoxic T lymphocytes.

The molecular requirements for recognition of antigen-modified cells by cytotoxic T lymphocyte precursors (CTLp) and their activated progeny, cytotoxic T lymphocytes (CTL), have been compared using haptenated stimulator and target cells. The antigen density requirements of T cell recognition by fluorescein-specific CTLp and CTL derived both from naive mice and from animals previously primed in vivo were determined. The cell surface hapten concentration required to stimulate CTLp cannot be distinguished from that required on target cells for lysis by their mature daughter CTL 5-7 days later. However, if the CTL (and their precursor CTLp) are derived from mice primed in vivo with hapten-conjugated cells, they require lower cell surface hapten densities for recognition than do the analogous T cell populations from naive animals. Thus, the maturation of CTLp into CTL during 5-7 days in vitro does not result in any functionally relevant change in the nature or density of antigen receptors on the surface of the T cell. This is in contrast to the apparent selection which occurs over longer time periods in vivo following priming.

A recombinational hotspot at the triplo-lethal locus of Drosophila melanogaster.

In the genome of Drosophila melanogaster there is only one locus, Tpl, that is triplo-lethal; it is also haplo-lethal. Previous work has identified 3 hypomorphic alleles of Tpl which rescue animals carrying a duplication of Tpl, but which are not dominant lethals as null mutations or deficiencies would be. We have found that all three hypomorphic alleles act as site-specific hotspots for recombination when heterozygous with a wild-type homolog. Recombination between the flanking markers ri and Ki is increased 6.5-10.5-fold in the presence of Tpl hypomorphic alleles. The increased recombination was found to occur between Tpl and Ki, while recombination in other adjacent regions is unchanged. The use of isogenic Tpl+ controls, and the use of flanking intervals in the mutant chromosomes allows us to rule out the interchromosomal effect as a cause. We have also observed premeiotic recombination occurring at the Tpl hypomorphic alleles in male heterozygotes. We hypothesize that transposons are responsible for both the hypomorphic phenotype and the high frequency of recombination.

The kinetics of generation of influenza-specific cytotoxic T-lymphocyte precursor cells.

A dramatic increase in the number of restimulatable (memory) cytolytic T-cell precursors (CTLps) has been demonstrated to occur 2 to 4 days postimmunization. By 4 or at the latest 6 days following in vivo priming, the frequency of restimulatable influenza-specific CTLps has reached that characteristic of a memory response. This indicates that the proliferative events which give rise to memory CTLps occur earlier than might have been expected and in fact are completed prior to the cessation of othe primary CTL response. Such information may have important significance to future immunomodulatory efforts aimed at perturbing the establishment of T-cell memory in vivo.