We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

/pdf/construction-of-escherichia-coli-k-12-in-frame-single-gene-4c68fx6pb1.pdf

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

http://wolfson.huji.ac.il/expression/local/bacteria/studierAutoInduced.pdf

Protein production by auto-induction in high-density shaking cultures

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional ∼12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

/pdf/the-sequence-of-the-human-genome-1bb4y67cls.pdf

The Sequence of the Human Genome

Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation MiRs are transcribed as short hairpin precursors (≈70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL) Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority (≈68%) of CLL cases

https://www.pnas.org/content/pnas/99/24/15524.full.pdf

Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia

Apolipoprotein E, type epsilon 4 allele (APOE epsilon 4), is associated with late-onset familial Alzheimer's disease (AD). There is high avidity and specific binding of amyloid beta-peptide with the protein ApoE. To test the hypothesis that late-onset familial AD may represent the clustering of sporadic AD in families large enough to be studied, we extended the analyses of APOE alleles to several series of sporadic AD patients. APOE epsilon 4 is significantly associated with a series of probable sporadic AD patients (0.36 +/- 0.042, AD, versus 0.16 +/- 0.027, controls [allele frequency estimate +/- standard error], p = 0.00031). Spouse controls did not differ from CEPH grandparent controls from the Centre d'Etude du Polymorphisme Humain (CEPH) or from literature controls. A large combined series of autopsy-documented sporadic AD patients also demonstrated highly significant association with the APOE epsilon 4 allele (0.40 +/- 0.026, p < or = 0.00001). These data support the involvement of ApoE epsilon 4 in the pathogenesis of late-onset familial and sporadic AD. ApoE isoforms may play an important role in the metabolism of beta-peptide, and APOE epsilon 4 may operate as a susceptibility gene (risk factor) for the clinical expression of AD.

https://n.neurology.org/content/neurology/43/8/1467.full.pdf

Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease.

Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

Molecular cloning : a laboratory manual

Purification of nucleic acids by extraction with phenol:chloroform.

1. Using Plasmid Vectors in Molecular Cloning 2. Bacteriophage bb and Its Vectors 3. Working with Bacteriophage M13 Vectors 4. Working with High-capacity Vectors 5. Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis 6. Preparation and Analysis of Eukaryotic Genomic DNA 7. Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells 8. In Vitro Amplification of DNA by the Polymerase Chain Reaction 9. Preparation of Radiolabeled DNA and RNA Probes 10. Working with Synthetic Oligonucleotide Probes 11. Preparation of cDNA Libraries and Gene Identification 12. DNA Sequencing 13. Mutagenesis 14. Screening Expression Libraries 15. Expression of Cloned Genes in Escherichia coli 16. Introducing Cloned Genes into Cultured Mammalian Cells 17. Analysis of Gene Expression in Mammalian Cells 18. Protein Interaction Technologies Appendices Index

The Condensed Protocols from Molecular Cloning: A Laboratory Manual

INTRODUCTIONThis protocol describes the separation of proteins by SDS-polyacrylamide gel electrophoresis. SDS is used with a reducing agent and heat to dissociate the proteins. SDS-polypeptide complexes form and migrate through the gels according to the size of the polypeptide. By using markers of known molecular weight, the molecular weight of the polypeptide chain(s) can be estimated.

SDS-Polyacrylamide Gel Electrophoresis of Proteins.

Directional cloning into plasmid vectors.

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