Germ Line Transformation Experiments Research Articles

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BioTechniquesVol. 56, No. 3 BioSpotlightOpen AccessBioSpotlightNathan BlowNathan BlowSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114140AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Bridging the gap in chromatin analysisDetermining the structure and organization of chromatin within cells is important for understanding how gene expression is regulated and how genomes evolve. While a number of techniques, including fluorescence in situ hybridization (FISH) and various chromatin conformation capture analyses (e.g., 3C and Hi-C), have been developed to study chromatin organization and interactions, each of these approaches has drawbacks. While FISH allows single-cell studies of chromatin interactions, the resolution of this technique can be limited to the diffraction limit of light, ∼250 nm. Super resolution microscopy techniques have improved upon this limitation, but still not to sufficient resolution to discern specific chromatin features or enable three-dimensional reconstruction of chromatin interactions. Chromatin conformation capture techniques enable much higher resolution studies of chromatin interactions but also require large numbers of cells, resulting in an inability to assess the frequencies of specific interactions within individual cells. In an effort to bridge the gap between the high resolution of chromatin conformation analysis and the single cell perspective provided by FISH, Chen et al. (Karolinska Institute, Stockholm, Sweden) present a new technique, chromatin in situ proximity (ChrISP), in the current issue of BioTechniques. The methodological basis of ChrISP lies in the well-developed in situ proximity ligation assay (ISPLA), wherein two probes create a fluorescent amplification signal when in close proximity to one another. Two forms of ChrISP were described by the authors: In one, rolling circle amplification (RCA) is used to amplify the signal between the two probes, while the second avoids RCA, instead relying on labeled “splinter” and “backbone” oligos to create a signal between the two chromatin probes. The authors demonstrate that by using the “Green-Splinter ChrISP” (splinter plus backbone oligos with no RCA) they were able to obtain resolutions greater than 17 nm in all 3 dimensions, a 30-fold improvement in the Z-plane in comparison to the other approaches. In the end, given the versatility and simplicity of the ChrISP technique, Chen et al. predict that the method will prove extremely useful when examining chromatin structure alone as well as the alignment of chromatin with other cell landmarks, such as the nuclear membrane and nucleoli.See “Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at high resolution”Needle-ing butterfliesAfter years of development and optimization, it is easy for Drosophila researchers to take germline transformation for granted. While it is a demanding technique that quite often requires a large number of injections for success, a transformant can usually be obtained. For other species though, the tools for germline transformation are less well developed, presenting a challenge for scientists wanting to assess genotype/phenotype relationships. But in this issue of BioTechniques, a team of researchers led by Jeffrey Marcus (University of Manitoba, Winnipeg, Canada) report on an improved injection needle design that significantly increases transformation efficiencies and experimental outcomes when working with the buckeye butterfly, Junonia coenia. In pilot injections using a non-optimized needle design previously used for germline transformation in another butterfly species, the authors observed 100% mortality of injected Junonia ova. In an effort to find a needle that would work for Junonia transformation, the group took inspiration from standard Drosophila needles, which have a steep taper and large orifice. Employing these “new” needle designs while injecting Junonia ova resulted in a significantly improved outcome: 21.7% of injected ova hatched with a transformation rate of 3%. Marcus and his team next checked whether or not their new design also showed increased transformation efficiency with other butterfly and moth species beyond Junonia—finding that these needles were indeed more efficient than previous designs. The new needles should be a welcome advance for the large number of researchers working on lepidopteran species where higher transformation efficiency is especially important since fewer ova are available, making genetic analysis a more realistic possibility.See “Improved injection needles facilitate germline transformation of the buckeye butterfly Junonia coenia”A new needle design (Needle type 2) for ova injection during germline transformation experiments of butterfly and moth species significantly improved transformation efficiencies.FiguresReferencesRelatedDetails Vol. 56, No. 3 Follow us on social media for the latest updates Metrics Downloaded 122 times History Published online 3 April 2018 Published in print March 2014 Information© 2014 Author(s)PDF download

Transcriptional Specificity of Drosophila Dysfusion and the Control of Tracheal Fusion Cell Gene Expression

The Drosophila Dysfusion basic-helix-loop-helix-PAS (bHLH-PAS) protein controls the transcription of genes that mediate tracheal fusion. Dysfusion is highly related to the mammalian Nxf protein that has been implicated in nervous system gene regulation. Toward the goal of understanding how Dysfusion controls fusion cell gene expression, the biochemical properties of Dysfusion were investigated using protein interaction experiments, cell culture-based transcription assays, and in vivo transgenic analyses. Dysfusion dimerizes with the Tango bHLH-PAS protein, and together they act as a DNA binding transcriptional activator. Dysfusion/Tango binds multiple NCGTG binding sites, with the following preference: TCGTG > GCGTG > ACGTG > CCGTG. This binding site promiscuity differs from the restricted binding site preferences of other bHLH-PAS/Tango heterodimers. However, it is identical to the binding site preferences of mammalian Nxf/Arnt, indicating that the specificity is evolutionarily conserved. Germ line transformation experiments using a fragment of the CG13196 Dysfusion target gene allowed identification of a fusion cell enhancer. Experiments in which NCGTG sites were mutated individually and in combination revealed that TCGTG sites were required for fusion cell expression but that the single ACGTG and GCGTG sites present were not. Finally, a reporter transgene containing four tandemly arranged TCGTG elements has strong expression in tracheal fusion cells. Transgenic misexpression of dysfusion further revealed that Dysfusion has the ability to activate transcription in multiple cell types, although it does this most effectively in tracheal cells and can only function at mid-embryogenesis and later.

Isolation and characterization of the Xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata.

Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated in many species representing a broad cross section of the major groups of living organisms, including the cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies.

Highly sensitive, fluorescent transformation marker for Drosophila transgenesis.

The efficiency of transposon-mediated germline transformation is dependent on the transposon mobility in the host embryo, and on the detectability of the used transformation marker. Therefore, high susceptibility of the transformation marker to position effect suppression is a disadvantage. Here we present data that the eye-specific expression of green fluorescent protein, driven by the 3xP3-EGFP marker, outperforms the commonly used "mini"-white transformation marker in Drosophila germline transformation experiments: 3xP3-EGFP is more sensitive than "mini"-white in identifying transgenic individuals and reacts differently to position effect suppression. Therefore, 3xP3-EGFP offers an ideal marker for applications in functional genomics where as many gene loci as possible should be targeted in the genome of a specific organism, for example, as intended in the Drosophila gene disruption project. Furthermore, we give a detailed description of the embryonic and larval expression mediated by the 3xP3-EGFP marker. These pre-adult expression patterns, and the potentially universal applicability of the transformation marker also offer additional advantages for selecting transgenic individuals in organisms other than Drosophila. This will be of great interest to the field of evolutionary developmental biology and to modern pest management programs.

A versatile vector set for animal transgenesis.

Genetic manipulation of a series of diverged arthropods is a highly desirable goal for a better understanding of developmental and evolutionary processes. A major obstacle so far has been the difficulty in obtaining marker genes that allow easy and reliable identification of transgenic animals. Here, we present a versatile vector set for germline transformation based on the promiscuous transposons mariner, Hermes and piggyBac. Into these vectors, we introduced a potentially universal marker system that is comprised of an artificial promoter containing three Pax-6 homodimer binding sites. This promoter drives strong expression of spectral variants of the enhanced green fluorescent protein (EGFP) in larval, pupal, and adult photoreceptors. Using special filter sets, the yellow (EYFP) and cyan (ECFP) variant are fully distinguishable and therefore represent a separable pair of markers. Furthermore, we adapted a simple plasmid-based transposition assay system to enable quick functional tests of our vectors in different arthropod species before employing them in more laborious germline transformation experiments. Using this system we demonstrate that our vectors transpose in both Drosophila melanogaster and Drosophila virilis.

Let-756, a C. elegans fgf essential for worm development.

In vertebrates, Fibroblast Growth Factors (FGFs) and their receptors are involved in various developmental and pathological processes, including neoplasia. The number of FGFs and their large range of activities have made the understanding of their precise functions difficult. Investigating their biology in other species might be enlightening. A sequence encoding a putative protein presenting 30-40% identity with the conserved core of vertebrate FGFs has been identified by the C. elegans sequencing consortium. We show here that this gene is transcribed and encodes a putative protein of 425 amino acids (aa). The gene is expressed at all stages of development beyond late embryogenesis, peaking at the larval stages. Loss-of-function mutants of the let-756 gene are rescued by the wild type fgf gene in germline transformation experiments. Two partial loss-of-function alleles, s2613 and s2809, have a mutation that replaces aa 317 by a stop. The truncated protein retains the FGF core but lacks a C-termins portion. These worms are small and develop slowly into clear and scrawny, yet viable and fertile adults. A third allele, s2887, is inactivated by an inversion that disrupts the first exon. It causes a developmental arrest early in the larval stages. Thus, in contrast to the other nematode fgf gene egl-17, let-756/fgf is essential for worm development.

Regulatory elements in the promoter of the vitelline membrane gene VM32E of Drosophila melanogaster direct gene expression in distinct domains of the follicular epithelium.

The Drosophila vitelline membrane protein gene VM32E is expressed according to a precise temporal and spatial program in the follicle cells. Results from germ line transformation experiments using different fragments of the -465/-39 VM32E region fused to the hsp/lacZ reporter gene revealed that the region -348/-39 is sufficient to confer the wild-type expression pattern. Within this segment, distinct cis-regulatory elements control VM32E expression in ventral and dorsal follicle cells. The region between -135/-113 is essential for expression of the VM32E gene in the ventral columnar follicle cells. Expression in the dorsal domain requires the two regions -348/-254 and -118/-39. Furthermore, the region -253/-119 appears to contain a negative element that represses gene activity in anterior centripetal cells. We suggest that the expression of the VM32E gene throughout the follicular epithelium is controlled by specific cis-regulatory elements acting in distinct spatial domains and following a precise developmental program.

Characterization of the EstP protein in Drosophila melanogaster and its conservation in drosophilids.

The beta-esterase cluster of D. melanogaster comprises two tandemly duplicated genes. Est6 encodes the well-characterized 5' gene, but the product of the second gene, denoted EstP, had not previously been identified. Here we show that the EstP gene encodes the carboxylesterase EST7. Expression of EstP using the Baculovirus system led to production of a carboxylesterase biochemically indistinguishable from EST7. Furthermore, a naturally occurring EstP variant produces greatly reduced amounts of EstP mRNA and no detectable EST7 protein. Finally, introduction of a wild-type copy of EstP by germline transformation into the variant strain confers the wild-type EST7 phenotype. We show that EST7 differs from EST6 in its substrate and inhibitor specificities and tissue distribution. Germline transformation experiments show that EstP expression is controlled by sequences located between 192 bp 5' and 609 bp 3' of the EstP coding region. Data comparisons with other drosophilid esterases suggest that the site of expression and hence the function, of EST7 has been conserved across lineages in both the subgenera Drosophila and Sophophora.

Mosaic analysis using a ncl-1 (+) extrachromosomal array reveals that lin-31 acts in the Pn.p cells during Caenorhabditis elegans vulval development.

We describe a genetic mosaic analysis procedure in which Caenorhabditis elegans mosaics are generated by spontaneous loss of an extrachromosomal array. This technique allows almost any C. elegans gene that can be used in germline transformation experiments to be used in mosaic analysis experiments. We identified a cosmid clone that rescues the mutant phenotype of ncl-1, so that this cell-autonomous marker could be used to analyze mosaic animals. To determine the sites of action for unc-29 and lin-31, an extrachromosomal array was constructed containing the ncl-1(+) cosmid linked to lin-31(+) and unc-29(+) cosmids. This array is mitotically unstable and can be lost to produce a clone of mutant cells. The specific cell division at which the extrachromosomal array had been lost was deduced by scoring the Ncl phenotypes of individual cells in genetic mosaics. The Unc-29 and Lin-31 phenotypes were then scored in these animals to determine in which cells these genes are required. This analysis showed that unc-29, which encodes a subunit of the acetylcholine receptor, acts in the body muscle cells. Furthermore, lin-31, which specifies cell fates during vulval induction and encodes a putative transcription factor similar to HNF-3/fork head, acts in the Pn.p cells.

Three protein binding sites form an enhancer that regulates sex- and fat body-specific transcription of Drosophila yolk protein genes.

Transcription of the Drosophila yolk protein (Yp) genes is regulated by the somatic sex determination pathway. A gene at the bottom of this pathway, doublesex, encodes the female-specific DSXF and male-specific DSXM proteins that bind to and regulate transcription from several sites in the Yp genes. We report site-directed mutagenesis, protein binding and germline transformation experiments that identify and characterize the activity of a single binding site (dsxA) for the doublesex proteins and two binding sites for other regulatory proteins. A single copy of the three sites is sufficient to direct the sex and fat body specificities of Yp transcription. The sites form an enhancer with two strongly synergistic enhancer elements. One element (22 bp) consists of dsxA and an overlapping site, bzip1, that binds the DmC/EBP (slbo) protein, a member of the bZIP family of transcriptional activators. The other element is an 11 bp binding site (ref1) for an unknown protein. Tissue-specific activation requires strong cooperation between the ref1 site and the bzip1 or dsxA sites. Sex specificity is regulated exclusively by the dsxA site which connects the sex determination pathway to the target gene through DSXM repression and DSXF activation.

Control of CNS midline transcription by asymmetric E-box-like elements: similarity to xenobiotic responsive regulation.

Central nervous system midline cells constitute a discrete group of Drosophila embryonic cells with numerous functional and developmental roles. Corresponding to their separate identity, the midline cells display patterns of gene expression distinct from the lateral central nervous system. A conserved 5 base pair sequence (ACGTG) was identified in central nervous system midline transcriptional enhancers of three genes. Germ-line transformation experiments indicate that this motif forms the core of an element required for central nervous system midline transcription. The central nervous system midline element is related to the mammalian xenobiotic response element, which regulates transcription of genes that metabolize aromatic hydrocarbons. These data suggest a model whereby related basic-helix-loop-helix-PAS proteins interact with asymmetric E-box-like target sequences to control these disparate processes.

Genetic Control of Programmed Cell Death in Drosophila

A gene, reaper (rpr), that appears to play a central control function for the initiation of programmed cell death (apoptosis) in Drosophila was identified. Virtually all programmed cell death that normally occurs during Drosophila embryogenesis was blocked in embryos homozygous for a small deletion that includes the reaper gene. Mutant embryos contained many extra cells and failed to hatch, but many other aspects of development appeared quite normal. Deletions that include reaper also protected embryos from apoptosis caused by x-irradiation and developmental defects. However, high doses of x-rays induced some apoptosis in mutant embryos, and the resulting corpses were phagocytosed by macrophages. These data suggest that the basic cell death program is intact although it was not activated in mutant embryos. The DNA encompassed by the deletion was cloned and the reaper gene was identified on the basis of the ability of cloned DNA to restore apoptosis to cell death defective embryos in germ line transformation experiments. The reaper gene appears to encode a small peptide that shows no homology to known proteins, and reaper messenger RNA is expressed in cells destined to undergo apoptosis.

Transcriptional activation domains of the single-minded bHLH protein are required for CNS midline cell development

The single-minded gene functions as a master developmental regulator within the midline cell lineage of the embryonic central nervous system of Drosophila melanogaster. Genetic experiments suggest that Single-minded can function as a transcriptional activator. Regions of the Single-minded protein were fused to the DNA binding domain of the mammalian transcription factor Sp1 and shown to activate transcription from a reporter gene linked to Sp1 binding sites. Three independent activation domains were identified in the carboxy terminal region of Single-minded that include areas rich in serine, threonine, glutamine and proline residues. Germ line transformation experiments indicate that the carboxy terminal activation domains, the PAS dimerization domain, and the putative DNA binding basic domain of Single-minded are required for expression of CNS midline genes in vivo. These results define in vivo a functional activation domain within Single-minded and suggest a model in which Single-minded activates transcription through a direct interaction with promoter elements of CNS midline genes.

Control of expression of the homeotic labial (lab) locus of Drosophila melanogaster. evidence for both positive and negative autogenous regulation

The homeotic gene labial (lab) is required for proper development of the embryonic and adult head in Drosophila melanogaster. The lab gene product accumulates in a complex pattern in both embryonic and imaginal tissue. During embryogenesis, lab is expressed in the endodermally derived cells of the midgut, in ectodermally derived cells of the procephalon and dorsal ridge, and in a small subset of progenitor sensory cells. Imaginal expression is restricted to a narrow region of the peripodial membrane of the eye-antennal disc. As part of our continuing effort to understand the role of lab in development, we have begun a dissection of the regulatory elements of the lab transcription unit and used germ line transformation experiments to determine which aspects of the observed expression pattern are essential for proper head development and viability. Transgenic embryos harboring an abridged lab gene are able to overcome the embryonic lethality associated with the loss of lab function and survive to adulthood. Interestingly, in these transgenic lines the lab protein accumulates only in a subset of those embryonic cells that normally express the gene, namely the procephalon and the anterior midgut. We also find that, once initiated, lab expression is maintained by positive autoregulation. Although lab minigene activity is sufficient to rescue the embryonic lethality of lab mutations, the transgenes fail to rescue defects in the adult head capsule. However, the defects observed in this study encompass a broader domain than those seen using somatic recombination to generate lab- clonal tissue. The failed rescue and observed cuticular defects are, at least in part, explained by the observation that the transgenes, rather than failing to be expressed, are associated with ectopic accumulation of lab protein in the peripodial membrane of the antennal disc. Moreover, this aberrant expression pattern is correlated with the abnormal expression of two other homeotic genes, Deformed (Dfd) and Sex combs reduced (Scr) in the eye-antennal disc. These results are only observed when the transgene is resident in a lab- genotype and ectopic expression of lab and misregulation of Dfd and Scr are not seen in a lab+ background. This result suggests that the wild-type lab gene product is necessary for the normal regulation of the locus in the imaginal discs, but unlike the case in the embryo, the event is negative. We discuss the biological implications of these results in relation to the role of lab in development.

Chromosome tangling and breakage at anaphase result from mutations in lodestar, a Drosophila gene encoding a putative nucleoside triphosphate-binding protein.

We describe a Drosophila maternal-effect gene, lodestar, mutations in which cause chromatin bridges at anaphase. lodestar maps to cytological position 84D13-14, and we identified the lodestar gene in germ-line transformation experiments by the ability of a genomic fragment to restore fertility to females homozygous for lodestar mutations. lodestar encodes a potential nucleoside triphosphate binding protein, which is a novel member of the D-E-A-H box family of proteins. Antibodies raised against the lodestar gene product detect a protein that undergoes cell cycle-dependent changes in distribution in the embryo. The protein is cytoplasmic at interphase, and rapidly enters the nucleus early in prophase. It is restricted to the region enclosed by the spindle envelope during metaphase and anaphase; but by telophase, the lodestar protein is contained entirely within the reforming nucleus.

Autoregulation of the splicing of transcripts from the transformer-2 gene of Drosophila.

The Drosophila transformer-2 gene uses alternative promoters and splicing patterns to generate four different mRNAs that together encode three putative RNA-binding polypeptides. The transformer-2 products expressed in somatic tissues function to regulate the RNA splicing of the sex determination gene doublesex, whereas products expressed in the male germ line play an unknown, but essential, role in spermatogenesis. Two alternatively spliced transformer-2 transcripts, each encoding a different putative RNA-binding protein, are found only in the male germ line. These male germ line-specific mRNAs differ from each other by the presence or absence of a single intron called M1. We show that M1-containing transcripts make up a majority of transformer-2 germ-line transcripts in wild-type males but fail to accumulate in males homozygous for transformer-2 null mutations. Germ-line transformation experiments using a variety of reporter gene constructs demonstrate that specific polypeptide products of the transformer-2 gene itself normally repress M1 splicing in the male germ line. Thus, in addition to its role in the sex-specific control of doublesex RNA splicing in somatic tissues, the transformer-2 gene also regulates the splicing of its own transcripts in the male germ line. We propose that this autoregulatory function may serve in negative feedback control of transformer-2 activity during spermatogenesis. The finding that transformer-2 controls multiple splicing decisions suggests that a variety of different alternative splicing choices could be regulated by a relatively limited number of trans-acting factors.

Molecular Transfer of a Species-specific Behavior from Drosophila simulans to Drosophila melanogaster

Drosophila males modulate the interpulse intervals produced during their courtship songs. These song cycles, which are altered by mutations in the clock gene period, exhibit a species-specific variation that facilitates mating. We have used chimeric period gene constructs from Drosophila melanogaster and Drosophila simulans in germline transformation experiments to map the genetic control of their song rhythm difference to a small segment of the amino acid encoding information within this gene.

Further sequence requirements for male germ cell-specific expression under the control of the 14 bp promoter element (β2UE1) of theDrosophilaβ2 tubulin gene

We have investigated a 14 bp promoter element (beta 2UE1) that is required for testis-specific expression of the Drosophila beta 2 tubulin gene. To further elucidate the role of the 14 bp element, we fused different promoter constructs to the E. coli lacZ gene and established transgenic strains with the aid of the Drosophila P-element transformation system. Germ line transformation experiments with constructs in which the element in the beta 2 tubulin gene promoter was exchanged for a related sequence from the promoter region of the Drosophila beta 3 tubulin gene led to a dramatic reduction in the expression of the lacZ gene in the testis. Exchanging the 14 bp promoter element for a similar sequence from the distal promoter of the Drosophila alcohol dehydrogenase gene abolished expression. This might indicate that the sequence differences between the beta 2UE1 and the beta 2UE1-related elements reflect functional differences between these elements. Constructs in which the beta 2UE1 was fused to the hsp70 promoter revealed that testis-specific expression of a marker gene is obtained only when the element is located at the correct distance from the transcription initiation site. However, constructs in which the beta 2UE1 was inserted at about the correct position (between -41 and -54 bp) upstream of a truncated beta 3 tubulin gene promoter did not show any expression. By making beta 2-beta 3 gene promoter fusions it was found that both the region surrounding the beta 3 transcription initiation site as well as the first 116 b of beta 3 leader sequences independently reduce testis-specific expression. These findings suggest that the testis-specific expression of the Drosophila beta 2 tubulin gene underlies a unique regulatory mechanism.

The Caenorhabditis elegans gene lin-10 is broadly expressed while required specifically for the determination of vulval cell fates.

As a first step in a molecular dissection of the pathway controlling the determination of vulval cell fates in Caenorhabditis elegans, we have analyzed the gene lin-10. We show that loss-of-function mutations in this gene specifically prevent the induction of vulval cell lineages and result instead in the expression of hypodermal cell lineages. We isolated a transposon-insertion allele of lin-10 and used it to clone a genomic region that contains the lin-10 locus. The location of lin-10 within this region was determined by identifying a transcript affected by three independent lin-10 mutations and by delimiting the minimal segment of DNA sufficient to rescue the lin-10 mutant phenotype in germ line transformation experiments. The predicted lin-10 protein sequence is not similar to sequences in current data bases, suggesting that lin-10 defines a novel class of gene involved in the specification of cell fates. Although our genetic studies indicate that lin-10 is required specifically for the determination of vulval cell fates, lin-10 transcripts are present in cells other than vulval precursor cells. This result suggests that lin-10 may have a general but redundant role in development, functioning in diverse cell lineages to control cell fates. Alternatively, lin-10 may function specifically in vulval development, in which case lin-10 activity could be regulated at a post-transcriptional level or could have biological consequences only in conjunction with the products of other genes.

A novel spatial transcription pattern associated with the segmentation gene, giant, of Drosophila.

The segmentation gene, giant, is located in 3A1 within a cloned chromosome region surrounding the zeste locus. Rearrangement breakpoints associated with giant mutations were localized on the genomic clone map, and nearby transcription units were identified. One transcription unit is active during early embryogenesis and its transcripts are spatially localized from blastoderm into extended germband stages, consistent with expected expression patterns predicted by the 'gap' phenotype of giant mutants. Germ line transformation experiments using a 10-kb DNA fragment containing this transcription unit gave complete rescue of the abdominal giant defect but only partial correction of the head defect. The effect of mutations in three other gap loci, Kr, kni and hb, were also analyzed.