The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

https://njc.rockefeller.edu/pdf4/Class2-LeeAmbros1993.pdf

The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

The 2016 revision of the World Health Organization classification of lymphoid neoplasms

Methods of altering retroviral tropism have been discovered. Such methods are useful, e.g., for developing retroviral vectors for gene therapy.

Altering viral tropism

Ron is a human receptor for the macrophage-stim- ulating protein (MSP). Exon 11 skipping of Ron pre-mRNA produces the Ron∆165 protein that has a deletion of a 49 amino acid region in the β-chain extracellular domain. Ron∆165 is constitutively active even in the absence of its ligand. Through stepwise deletion analysis, we identified a 2-nt RNA enhancer, which is located 74 nt upstream from the 5' splice site of exon 11, for exon 11 inclusion. Through double-base and single-base substitution analysis of the 2-nt RNA, we demonstrated that the GA, CC, UG and AC dinucleotides on exon 11, in addition to the wild-type AG sequence, function as enhancers for exon 11 inclusion of the Ron pre-mRNA.

/pdf/a-2-nt-rna-enhancer-on-exon-11-promotes-exon-11-inclusion-of-1q3n4d7trh.pdf

A 2-nt RNA enhancer on exon 11 promotes exon 11 inclusion of the Ron proto-oncogene.

This protocol describes the use of a monophasic lysis reagent to isolate total RNA from mammalian cells (grown as either a monolayer or in suspension) or tissues. An alternative protocol for the isolation of RNA from a small quantity of cells (102-104) or tissue (1-10 mg) is also included. In the latter case, glycogen is added to the monophasic lysis reagent and is used as a carrier to increase the recovery of RNA.

/pdf/purification-of-total-rna-from-mammalian-cells-and-tissues-3ji2jhez9x.pdf

Purification of Total RNA from Mammalian Cells and Tissues

The Klenow fragment, which retains the template-dependent deoxynucleotide polymerizing activity and the 3' → 5' exonuclease of the holo-enzyme but lacks its powerful 5' → 3' exonuclease activity, is used to fill recessed 3' termini of dsDNA. In this protocol, fragments suitable as templates for the end-filling reaction are produced by digestion of DNA with an appropriate restriction enzyme. The Klenow enzyme is then used to catalyze the attachment of dNTPs to the recessed 3'-hydroxyl groups.

Labeling 3' Termini of Double-stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I.

The invention features TAF-47 and TAF-68, proteins associated with the TATA-box binding protein and the nucleic acid molecules encoding them These proteins are required for viability of S cerevisiase and activated in vitro transcription

Michael R. Green

Papers

Altering viral tropism

A 2-nt RNA enhancer on exon 11 promotes exon 11 inclusion of the Ron proto-oncogene.

Purification of Total RNA from Mammalian Cells and Tissues

Labeling 3' Termini of Double-stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I.

Novel tata-box binding protein-associated factors essential for yeast viability