A neurotransmitter transporter encoded by the Drosophila inebriated gene

Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl−-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron.

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: Are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.

Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.

We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.

Comparative mapping of Andropogoneae: Saccharum L. (sugarcane) and its relation to sorghum and maize

Comparative genetic maps of Papuan Saccharum officinarum L. (2n = 80) and S. robustum (2n = 80) were constructed by using single-dose DNA markers (SDMs). SDM-framework maps of S. officinarum and S. robustum were compared with genetic maps of sorghum and maize by way of anchor restriction fragment length polymorphism probes. The resulting comparisons showed striking colinearity between the sorghum and Saccharum genomes. There were no differences in marker order between S. officinarum and sorghum. Furthermore, there were no alterations in SDM order between S. officinarum and S. robustum. The S. officinarum and S. robustum maps also were compared with the map of the polysomic octoploid S. spontaneum ‘SES 208’ (2n = 64, x = 8), thus permitting relations to homology groups (“chromosomes”) of S. spontaneum to be studied. Investigation of transmission genetics in S. officinarum and S. robustum confirmed preliminary results that showed incomplete polysomy in these species. Because of incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. To coalesce S. officinarum and S. robustum linkage groups into homology groups (composed of homologous pairing partners), they were compared with sorghum (2n = 20), which functioned as a synthetic diploid. Groupings suggested by comparative mapping were found to be highly concordant with groupings based on highly polymorphic restriction fragment length polymorphism probes detecting multiple SDMs. The resulting comparative maps serve as bridges to allow information from one Andropogoneae to be used by another, for breeding, ecology, evolution, and molecular biology.

High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: A major fruit weight quantitative trait locus in tomato

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2. Using an F2 nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) × Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 ± 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be ≤ 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

Centromere mapping and orientation of the molecular linkage map of rice (Oryza sativa L.).

Rice has become a model cereal plant for molecular genetic research. Rice has the most comprehensive molecular linkage maps with more than 2000 DNA markers and shows synteny and colinearity with the maps of other cereal crops. Until now, however, no information was available about the positions of centromeres and arm locations of markers on the molecular linkage map. Secondary and telotrisomics were used to assign restriction fragment length polymorphism markers to specific chromosome arms and thereby to map the positions of centromeres. More than 170 restriction fragment length polymorphism markers were assigned to specific chromosome arms through gene dosage analysis using the secondary and telotrisomics and the centromere positions were mapped on all 12 linkage groups. The orientations of seven linkage groups were reversed to fit the "short arm on top" convention and the corrected map is presented.

Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member: comparative mapping of the locus for the human peptide antibiotic FALL-39.

PR-39 is a porcine 39-aa peptide antibiotic composed of 49% proline and 24% arginine, with an activity against Gram-negative bacteria comparable to that of tetracycline. In Escherichia coli, it inhibits DNA and protein synthesis. PR-39 was originally isolated from pig small intestine, but subsequent cDNA cloning showed that the gene is expressed in the bone marrow. The open reading frame of the clone showed that PR-39 is made as 173-aa precursor whose proregion belongs to the cathelin family. The PR39 gene, which is rather compact and spans only 1784 bp has now been sequenced. The coding information is split into four exons. The first exon contains the signal sequence of 29 residues and the first 37 residues of the cathelin propart. Exons 2 and 3 contain only cathelin information, while exon 4 codes for the four C-terminal cathelin residues and the mature PR-39 peptide extended by three residues. The sequenced upstream region (1183 bp) contains four potential recognition sites for NF-IL6 and three for APRF, transcription factors known to regulate genes for both cytokines and acute phase response factors. Genomic hybridizations revealed a fairly high level of restriction fragment length polymorphism and indicated that there are at least two copies of the PR39 gene in the pig genome. PR39 was mapped to pig chromosome 13 by linkage and in situ hybridization mapping. The gene for the human peptide antibiotic FALL-39 (also a member of the cathelin family) was mapped to human chromosome 3, which is homologous to pig chromosome 13.