Preparation of bacterial plasmid DNA.

The protocols in this unit describe methods for preparing bacterial plasmid DNA free from chromosomal DNA. The first is an alkaline lysis miniprep suitable for screening a moderate number of bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. The second is the first step to producing large amounts (milligrams) of plasmid DNA and is also based on alkaline lysis of the bacterial cells. The crude lysate generated in this protocol can be further purified by centrifugation using CsCl/ethidium bromide (CsCl/EtBr) equilibrium density gradients. Three support protocols provide information on how to grow overnight and larger cultures of bacteria, and how to monitor bacterial growth with a spectrophotometer. Other methods, some relying on commercially available ion-exchange columns, are discussed in the commentary.

Preparation of bacterial plasmid DNA.

The protocols in this unit describe methods for preparing bacterial plasmid DNA free from chromosomal DNA. The first is an alkaline lysis miniprep suitable for screening a moderate number of bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. The second is the first step to producing large amounts (milligrams) of plasmid DNA and is also based on alkaline lysis of the bacterial cells. The crude lysate generated in this protocol can be further purified by centrifugation using CsCl/ethidium bromide (CsCl/EtBr) equilibrium density gradients. Three support protocols provide information on how to grow overnight and larger cultures of bacteria, and how to monitor bacterial growth with a spectrophotometer. Other methods, some relying on commercially available ion-exchange columns, are discussed in the commentary.

Large-scale preparation of plasmid DNA.

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

Fasciclin II and Beaten path modulate intercellular adhesion in Drosophila larval visual organ development.

Previous studies demonstrated that Fasciclin II and Beaten path are necessary for regulating cell adhesion events that are important for motoneuron development in Drosophila. We observe that the cell adhesion molecule Fasciclin II and the secreted anti-adhesion molecule Beaten path have additional critical roles in the development of at least one set of sensory organs, the larval visual organs. Taken together, phenotypic analysis, genetic interactions, expression studies and rescue experiments suggest that, in normal development, secretion of Beaten path by cells of the optic lobes allows the Fasciclin II-expressing larval visual organ cells to detach from the optic lobes as a cohesive cell cluster. Our results also demonstrate that mechanisms guiding neuronal development may be shared between motoneurons and sensory organs, and provide evidence that titration of adhesion and anti-adhesion is critical for early steps in development of the larval visual system.

Genetic analysis of Drosophila larval optic nerve development.

To identify genes necessary for establishing connections in the Drosophila sensory nervous system, we designed a screen for mutations affecting development of the larval visual system. The larval visual system has a simple and stereotypic morphology, can be recognized histologically by a variety of techniques, and is unnecessary for viability. Therefore, it provides an opportunity to identify genes involved in all stages of development of a simple, specific neuronal connection. By direct observation of the larval visual system in mutant embryos, we identified 24 mutations affecting its development; 13 of these are larval visual system-specific. These 13 mutations can be grouped phenotypically into five classes based on their effects on location, path or morphology of the larval visual system nerves and organs. These mutants and phenotypic classifications provide a context for further analysis of neuronal development, pathfinding and target recognition.

DeLIMiting development.

LIM-homeodomain transcription factors (LIM-HD) regulate expression of genes that pattern the body and generate cell specificity during development in invertebrates and vertebrates. It is especially interesting that most are expressed in and participate in the development of the nervous system. LIM-HD proteins are themselves regulated by both intramolecular and intermolecular interactions mediated by the LIM domains. LIM domains positively regulate LIM-HD activity by promoting protein-protein interactions that allow cooperative binding to regulatory regions of tissue-specific promoters. They also negatively regulate LIM-HD activity, possibly by preventing HD association with DNA. Interaction of LIM domains with other proteins relieves this interference, permitting DNA binding and providing a mechanism for refining LIM-HD activity. The recurrence of LIM-HD proteins in fundamental developmental processes emphasizes the importance of their function and regulation and provides an opportunity to identify mechanisms and molecules underlying patterning and cell specification.

Arrowhead encodes a LIM homeodomain protein that distinguishes subsets of Drosophila imaginal cells.

The Arrowhead gene encodes a LIM-homeodomain transcription factor required for establishment of a subset of imaginal tissues: the abdominal histoblasts and the salivary gland imaginal rings. Consistent with its role in development, during embryogenesis Arrowhead is expressed in each abdominal segment and in the labial segment. Late in embryonic development, expression is refined to the abdominal histoblasts and salivary gland imaginal ring cells themselves. When ectopically expressed in imaginal disc cells, Arrowhead causes programmed cell death and loss of corresponding adult structures. Therefore, Arrowhead expression is required for development of one set of imaginal cells and is incompatible with development of another, emphasizing the specificity of Arrowhead and the sensitivity of different target cells to its expression. Loss-of-function mutations in Arrowhead affect conserved or invariant amino acids in the LIM- and homeo-domains demonstrating the importance of these residues in LIM homeodomain protein activity.

Establishment of Drosophila imaginal precursor cells is controlled by the Arrowhead gene.

Metamorphosis in Drosophila melanogaster requires synchronization of numerous developmental events that occur in isolated imaginal precursor tissues. The imaginal primordia are established during embryonic stages and are quiescent for much of larval life. The Arrowhead gene is necessary for establishment of proper numbers of cells within a subset of imaginal precursor tissues. Loss-of-function mutations in Arrowhead reduce the number of abdominal histoblasts and salivary gland imaginal ring cells before the proliferative stages of their development. The number of abdominal histoblasts in mutant animals is approximately half that of wild-type, as might result from failure of a single early division of these cells. A neomorphic Arrowhead allele results in the specific loss of the retinal precursors by the early third instar, before they have begun to differentiate. Since Arrowhead mutations affect only subsets of imaginal tissue, there must be distinctions in the developmental regulation of different imaginal precursors. Arrowhead may be part of a regulatory pathway responsible for establishing the proper number of abdominal histoblasts and salivary gland imaginal ring cells. The neomorphic Arrowhead allele, which may cause misexpression of the Arrowhead gene in the eye-antenna imaginal disc, interferes with the establishment or proliferation of retinal precursor cells.

Isolation and characterization of the disconnected gene of Drosophila melanogaster.

Mutations in the disco (disconnected) gene prevent the establishment of stable connections between the larval optic nerves, the Bolwig's nerves, and their target cells in the brain during embryonic development. The failure of this initial connection is associated with aberrant development of the optic lobes which are largely degenerate in the mutant adult fly. In order to understand the role of disco in establishing this connection, we isolated and characterized the disco gene. A 22 kb DNA fragment can completely rescue the mutant phenotype. A single transcript, 2.9 kb in length, is found in this region and is expressed throughout development of the fly. We determined the nucleotide sequence of the disco gene to be unique when compared with sequences in a number of databases. The predicted amino acid sequence contains a region with similarity to the consensus established for the zinc finger motif. Mobilization of a P-element inserted near the gene resulted in the deletion of the 5' end of the gene and produced flies indistinguishable from those carrying the disco allele.

T-cell gamma gene is allelically but not isotypically excluded and is not required in known functional T-cell subsets.

The T-cell gamma genes, structurally related to immunoglobulin genes and the T-cell antigen-receptor alpha- and beta-chain genes, undergo somatic rearrangement in T-lineage cells. However, the role of the T-cell gamma genes has not yet been determined. To determine the potential for gamma gene expression in a set of well-characterized, cloned T-cell lines, we cloned all of the rearranged gamma genes from each cell line. The genes were sequenced to determine if the junction of the variable and joining regions maintained the proper translational reading frame. We then attempted to correlate the presence of an in-frame gamma gene with a T-cell subset. We were unable to establish such a correlation. We found evidence, however, that allelic exclusion influences the rearrangement of the gamma gene. This is consistent with the idea that the gamma gene product participates in establishing a clonally diverse population of T cells recognizing a polymorphic ligand. Isotypic exclusion does not apply to the gamma gene, however, suggesting different roles for the different gamma gene isotypes.

Diversity of murine gamma genes and expression in fetal and adult T lymphocytes.

The search for the genes encoding the T-cell receptor alpha and chains revealed a third gene, T gamma (ref. 1), which shares with t T alpha (refs 2-7) and T beta (refs 8-15) genes a number of structure features, including somatic rearrangement during T-cell development. T gamma gene expression appears to be unnecessary in son mature T cells and is at its greatest in fetal thymocytes encouraging speculation that T gamma has a role in T-cell development and may be involved in the recognition of polymorphic major histocompatibility complex (MHC) products during thymic education. One argument against the participation of T gamma in such a process has been its apparently limited diversity, due to the small number of gene segments available for rearrangement. We here describe the identification of additional T gamma V-gene segments and demonstrate that they can be rearranged to previously identified J- and C-gene segments and are expressed in fetal thymocytes. In addition we describe a variety of patterns of T gamma mRNA processing which may be significant for T gamma gene regulation.

Expression of the T-cell-specific gamma gene is unnecessary in T cells recognizing class II MHC determinants.

Subtractive complementary DNA cloning combined with partial protein sequencing has allowed identification of the genes encoding the alpha and beta subunits of T-cell receptors. The subtractive cDNA library prepared from the cytotoxic T lymphocyte (Tc) clone 2C has been found to contain a third type of clone encoding the gamma chain. The gamma gene shares several features with the alpha and beta genes: (1) assembly from gene segments resembling immunoglobulin V, J and C (respectively variable, joining and constant region) DNA segments; (2) rearrangement and expression in T cells and not in B cells; (3) sequences reminiscent of transmembrane and intracytoplasmic regions of integral membrane proteins; (4) a cysteine residue at the position expected for an interchain disulphide bond. The alpha and beta genes are expressed at equivalent levels in both Tc cells and helper T cells (TH). The gamma gene, obtained from 2C, has been found to be expressed in all Tc cells studied. Here we present evidence that strongly suggests that TH cells do not require gamma gene expression.

Unusual organization and diversity of T-cell receptor alpha-chain genes.

T lymphocytes recognize cell-bound antigens in the molecular context of the self major histocompatibility complex (MHC) gene products through the surface T-cell receptor(s). The minimal component of the T-cell receptor is a heterodimer composed of alpha and beta subunits, each of relative molecular mass (Mr) approximately 45,000 (refs 1-3). Recently, complementary DNA clones encoding these subunits have been isolated and characterized along with that of a third subunit of unknown function, termed gamma (refs 4-9). These studies revealed a primary structure for each subunit that was clearly similar to that of immunoglobulin and indicated a somatic rearrangement of corresponding genes that are also immunoglobulin-like. Recently, the analysis of the sequence organization of the T-cell receptor beta-chain and T-cell-specific gamma-chain gene families has been reported. We now present an initial characterization of the murine T-cell receptor alpha-chain gene family, and conclude that although it is clearly related to the gene families encoding immunoglobulins, T-cell receptor beta-chains and also T-cell gamma-chains, it shows unique characteristics. There is only a single constant (C) region gene segment, which is an exceptionally large distance (approximately 20-40 kilobases (kb) in the cases studied here) from joining (J) gene segments. In addition, the J cluster and the variable (V) segment number seen to be very large. Finally, in the case studied here, a complete alpha-chain gene shows no somatic mutation and can be assembled directly from V alpha, J alpha and C alpha segments without inclusion of diversity (D alpha) segments.

Gene for the RNA polymerase sigma subunit mapped in Salmonella typhimurium and Escherichia coli by cloning and deletion.

The genes for the RNA polymerase sigma subunit (rpoD) and DNA primase (dnaG) of Salmonella typhimurium have been cloned into lambda vectors. Combined restriction, deletion and functional analysis of the cloned fragment allows us to map the genes precisely on the fragment, establishes the direction in which rpoD is transcribed, and reveals the existence of at least one new gene in the vicinity. A closely homologous, smaller fragment of Escherichia coli DNA, also cloned into lambda, contains rpoD and at least part of dnaG.

Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase.

A gene affecting the sigma subunit of DNA-dependent RNA polymerase is tightly linked to dnaG at 66 min on the Escherichia coli chromosome. In order to create an easily selectable marker in this region, we inserted transposon-10, which carries a gene determining resistance to tetracycline (tet) near 66 min, and the order tolC-dnaG-sigma-tet was determined. We used frequency of contransduction with tet as a criterion to screen a collection of spontaneous temperature-sensitive Escherichia coli mutants that might affect the sigma subunit. One such mutant was found to map at the sigma locus. The sigma subunit isolated from this mutant is unstable at 46 degrees C in vitro and has an altered electrophoretic mobility. The temperature sensitivity of RNA synthesis in this mutant indicates that most transcription in E. coli is sigma dependent.