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.

/pdf/initial-sequencing-and-analysis-of-the-human-genome-5dapk1rsx1.pdf

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 for inhibiting the transcription-enhancingmethods for inhibiting the transcription-enhancing activity of the x protein of hepatitis b virus activity of the x protein of hepatitis b virus

To generate polymerase chain reaction (PCR) products that can be directionally cloned into a vector, different restriction sites are built into the forward and reverse primers that are used in the PCR. After PCR, the amplified product is purified, cleaved with the appropriate restriction enzymes, ligated into a vector with compatible cohesive ends, and used to transform E. coli.

Cloning Polymerase Chain Reaction Products: Addition of Restriction Sites to the Termini of Amplified DNA.

The disclosure relates to methods and compositions for regulating expression of DUX4. In some aspects, methods described by the disclosure are useful for treating a disease associated with aberrant DUX4 expression ( e.g ., facioscapulohumeral muscular dystrophy).

Therapeutic targets for facioscapulohumeral muscular dystrophy

Dot blotting and slot blotting are techniques for immobilizing several preparations of nucleic acids on the same solid support, usually a charged nylon membrane. The concentrations of the target sequence of interest can be estimated by hybridizing the immobilized samples to an appropriate probe. The amounts of target sequence are estimated by comparing the intensity of signals emitted by dots containing the test samples with standards containing known concentrations of the target sequence. This protocol describes the blotting and subsequent hybridization of RNA that has been purified from cells or tissues.

Dot and Slot Hybridization of Purified RNA.

Thin (0.4-1.5 mm) polyacrylamide-urea gels provide high resolution of RNAs up to 1000 nt in size and are capable of resolving single-stranded fragments of RNA that differ in length by as little as 1 nt. The polyacrylamide gel is cast between two glass plates that are separated by two thin Teflon or nylon spacers. A so-called shark's tooth comb or, less frequently, a standard slotted comb forms the sample wells into which the RNA samples are loaded before electrophoresis. In contrast to electrophoresis using agarose gels, which occurs while the gel is horizontal, polyacrylamide gels are run while in the vertical position. Gels are also typically run at 45°C-55°C, which is the melting temperature of RNA, and in the presence of 6-8 m urea. The gel recipe and protocol presented here for 8 m urea/TBE polyacrylamide gels can be used for a variety of applications including mapping RNA with nuclease S1, ribonuclease protection assay, or analysis of RNA by primer extension.

/pdf/separation-of-rna-according-to-size-electrophoresis-of-rna-1byuw94j5x.pdf

Michael R. Green

Papers

Methods for inhibiting the transcription-enhancingmethods for inhibiting the transcription-enhancing activity of the x protein of hepatitis b virus activity of the x protein of hepatitis b virus

Cloning Polymerase Chain Reaction Products: Addition of Restriction Sites to the Termini of Amplified DNA.

Therapeutic targets for facioscapulohumeral muscular dystrophy

Dot and Slot Hybridization of Purified RNA.

Separation of RNA according to Size: Electrophoresis of RNA through Denaturing Urea Polyacrylamide Gels.