A His2AvDGFP Fusion Gene Complements a Lethal His2AvD Mutant Allele and Provides an in Vivo Marker for Drosophila Chromosome Behavior

Abstract

We have generated Drosophila melanogaster lines carrying a modified genomic fragment which encodes the D. melanogaster variant H2A.F/Z class histone, His2AvD, fused to the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. We show here that the fusion protein consists of functional GFP and functional histone His2AvD. The His2AvD portion of the fusion gene was shown to be functional by rescue of His2AvD mutant lethality. Fluorescence of the fusion protein in vivo was observed in embryonic cleavage stage interphase nuclei and on chromosomes as early as cycle 9, correlating with activation of transcription. Unlike transcription factors, the His2AvDGFP protein remained on transcriptionally inactive chromosomes throughout mitosis. Subsequently, fluorescence was observed in nuclei at all stages of embryonic and larval development and in adult somatic tissues, consistent with the distribution of His2AvD observed by immunohistochemical staining. This functional fusion histone acts as an excellent in vivo marker for chromosomes and chromosome behavior and, given the ability of the fusion gene to prevent null-mutant lethality, without disrupting normal cellular functions. The very high level of conservation of the H2A.F/Z family of variant histones suggests that the equivalent fusion protein construct should function equally well in a wide range of organisms.

CENP-meta, an Essential Kinetochore Kinesin Required for the Maintenance of Metaphase Chromosome Alignment in Drosophila

“…To examine more directly how the absence of CENP-meta affects chromosome movement, chromosomes in CENP-meta deficient embryos were marked by introducing a stably inherited histone-2A-variant-GFP (Clarkson and Saint 1999). Real-time images of these embryos (videos available at http://www.jcb.org/cgi/content/full/150/1/1/DC1) revealed that congression of the fluorescently marked chromosomes appears to proceed relatively normally in cmet lx1 mutant embryos, but the chromosomes do not stably persist at the metaphase plate.…”

“…Germ-line clones were induced using FRT-ovoD1 and hsFLPase following Chou and Perrimon 1996. Clones containing chromosomes illuminated by histone-2A-var-GFP (a kind gift of Rob Saint, University of Adelaide; Clarkson and Saint 1999) were generated using FRT-cmet/SM1;hisvar-GFP/TM6B and hsFLPase/Y, FRT-ovoD1/CyO. Real-time movies were made using manually dechorionated embryos in heptane glue (Santamaria, 1986).…”

Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes

“…The GAL4-e33c upstream activating sequence (UAS)-gfp strain was generated by crossing flies carrying the GAL4-e33c enhancer trap (27) to flies carrying the gfp transgene under control of the UAS (GAL4 response element), thus achieving constitutive GFP expression in hemolymph and lymph glands hemocytes. For in vivo transfers, we used two GFP-expressing lines: His::GFP [ubiquitous expression of a fusion protein between histone His2AvD and GFP (28)] and Tum-l; His::GFP (generated by standard crossing). Stocks were fed standard cornmeal, molasses, yeast, and agar medium and were maintained at 25°C.…”

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

“…To examine the dynamics of Nup107 during mitosis, we first followed by time-lapse scanning confocal microscopy (TLSCM) the last three syncytial mitotic cycles (cycles 10 -13) of early Drosophila embryos expressing mRFP-Nup107 together with the GFP-tagged histone H2A variant His2AvD (Clarkson and Saint, 1999). Representative panels from one cleavage cycle (cycle 12) are shown in Figure 2A (Supplemental Movie 1).…”