Genetic and Phenotypic Analysis of Thirteen Essential Genes in Cytological Interval 22f1-2; 23b1-2 Reveals Novel Genes Required for Neural Development in Drosophila

In an attempt to identify mutations in the Drosophila synaptotagmin gene we have isolated many new rearrangements, point mutations and P element insertions in the 22F1-2; 23B1-2 cytological interval on chromosome arm 2L. This interval encompasses 13 cytological bands and is shown to contain 13 essential complementation groups, including decapentaplegic, synaptotagmin and Curly. Through chemical and P element mutagenesis we have isolated seven new deletions, which combined with previously isolated rearrangements, have allowed us to order most genes in the interval. A genomic walk covering approximately 100 kb within this interval spans at least five essential genes as identified by chromosomal aberrations. Preliminary phenotypic characterizations of the mutant phenotype and lethal phase is presented for many mutations. Three loci within this interval are shown to be required for proper neural development. Given that the average number of alleles per complementation group is greater than seven, it is very likely that all essential genes within this cytological interval have been identified.     

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.     

Genetic and molecular analysis of fs(1)h, a maternal effect homeotic gene in Drosophila

Mutations at the Drosophila melanogaster locus female sterile (1) homeotic (fs(1)h) result in segmental abnormalities including missing organs and homeotic transformations in the progeny of mutant mothers. Homeotic transformations are enhanced when the zygotes carry one of several third chromosome mutations, specifically alleles or deficiencies of the trithorax (trx) locus, also called Regulator-of-bithorax, and some alleles of bithorax complex (BX-C) genes. These observations suggest that maternally derived fs(1)h+ product is required, in interaction with trx and BX-C genes, for normal segment specification. The fs(1)h gene and an adjacent gene, lethal (1) myospheroid (l(1)mys), have been cloned by chromosomal walking. Mutations of fs(1)h were found within a 13-kb stretch of DNA. Poly(A)+ RNAs migrating as a doublet at 7.6 kb and a single band at 5.9 kb, which are homologous to the fs(1)h+ chromosomal region, are found in ovaries and early embryos. The largest RNAs are derived from a 20-kb chromosomal region encompassing the sites of all mapped fs(1)h alleles.     

Genomic and cDNA clones of the homeotic locus Antennapedia in Drosophila.

Homeotic genes are involved in the control of developmental pathways: dominant mutations at the Antennapedia locus of Drosophila, for example, lead to replacement of the antennae on the head of the fly by mesothoracic legs. Using a combination of chromosome walking and jumping, we have cloned a DNA region from Drosophila containing Antennapedia. Five DNA inversion rearrangements which are associated with the Antennapedia mutant phenotype were localized within a 25-kb region. Genomic DNA sequences from this area were used as hybridization probes to screen cDNA libraries prepared from Drosophila embryonic and pupal poly(A)+ RNA. A 2.2-kb cDNA sequence (903) was isolated which appears to derive from at least four non-contiguous chromosomal regions that span 100 kb. It includes the positions of the inversion breakpoints. A second cDNA of 2.9 kb (909) is composed of sequences from at least three chromosomal regions, two of which are similar or identical to sequences contained in the 903 clone but the third is derived from genomic DNA within a putative 903 intron. The unusual size and complexity of this locus are discussed.     

Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double-strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single-guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.     

Genetic and Molecular Mapping of Chromosome Region 85a in Drosophila Melanogaster

Chromosome region 85A contains at least 12 genetic complementation groups, including the genes dhod, pink and hunchback. In order to better understand the organization of this chromosomal segment and to permit molecular studies of these genes, we have carried out a genetic analysis coupled with a chromosome walk to isolate the DNA containing these genes. Complementation tests with chromosomal deficiencies permitted unambiguous ordering of most of the complementation groups identified within the 85A region. Recombinant bacteriophage clones were isolated that collectively span over 120 kb of 85A DNA and these were used to produce a molecular map of the region. The breakpoint sites of a number of 85A chromosome rearrangements were localized on the molecular map, thereby delimiting regions of the DNA that contain the various genetic complementation groups.