There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality.

Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

I and i

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

/pdf/multiplex-genome-engineering-using-crispr-cas-systems-2qm4sjme5b.pdf

Multiplex Genome Engineering Using CRISPR/Cas Systems

BACKGROUND Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

/pdf/activating-mutations-in-the-epidermal-growth-factor-receptor-4a7r2t8eq0.pdf

Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib

Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Chromatin, the physiological template of all eukaryotic genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA. Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a “histone code” that considerably extends the information potential of the genetic code. We propose that this epigenetic marking system represents a fundamental regulatory mechanism that has an impact on most, if not all, chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development.

/pdf/translating-the-histone-code-l291mddcr9.pdf

Translating the Histone Code

Only 1.2 kilobases (kb) at the 5' end of the 3.9-kb v-abl sequence in Abelson murine leukemia virus is required for fibroblast transformation. A precise delineation of this minimum transforming region was made by using small 5' or 3' deletions. Insertions of four amino acids, generated by putting synthetic DNA linkers into various restriction enzyme cleavage sites, abolished transforming activity, indicating that much of the internal sequence of the minimum transforming region plays a critical role in the transformation process. This 5' 1.2 kb of v-abl encodes protein-tyrosine kinase activity when expressed in Escherichia coli. Each of the mutations which caused a loss of transformation activity also resulted in a loss of protein-tyrosine kinase activity when expressed in E. coli. The minimum transforming region of v-abl contains amino acid homology to other protein-tyrosine kinase oncogenes, and a comparison with these oncogenes is presented.

The minimum transforming region of v-abl is the segment encoding protein-tyrosine kinase.

Ordinarily, sedimentation analysis of RNA preparations is carried out in simple aqueous solutions. Recently, numerous investigators have carried out experiments where RNA's in cytoplasmic extracts from various types of cells have been analyzed by sedimentation through gradients of sucrose. The implicit assumption has been made that the presence of the cytoplasmic extract does not in itself affect the rate of sedimentation of the RNA. However, we have recently found that some component of HeLa cell cytoplasmic extracts causes a 1.5-2-fold increase in the sedimentation rate of a number of species of RNA. Since this result bears on the interpretation of many types of experiments, we will report the phenomenon at this time, even though we know little about its mechanism.

https://www.pnas.org/content/pnas/56/3/999.full.pdf

The effect of HeLa cell cytoplasm on the rate of sedimentation of RNA

OVERVIEW A central class of transcriptional regulatory proteins are those with an apparent helix-loop-helix (HLH) dimerization motif abutting a DNA-binding basic region. The HLH region allows for homodimerization but, more importantly, various HLH proteins can interact to form heterodimers. The heterodimers appear to be major players in determining the expression of differentiated cellular functions in various differentiated cell types. INTRODUCTION Control of initiation of mRNA synthesis involves the interaction of DNA-binding proteins with each other and with specific DNA sequence elements. Families of such DNA-binding proteins have been identified that are structurally related. Among these families are proteins containing a zinc finger domain, a POU/homeobox domain, and leucine zipper dimerization domains. We have recently identified a new family of proteins that have in common a conserved DNA-binding and dimerization domain, designated the HLH motif. The size of the known HLH family is large (>30 members) and growing. Protein-protein interactions can occur between various HLH members, forming hetero-oligomers. The formation of hetero-oligomers and the large size of the HLH protein family offer an enormous scope of complexity and regulation. The purpose of this chapter is to survey HLH proteins and discuss their role in development. E-BOX REGULATORY ELEMENTS A wide variety of regulatory elements have been identified in the past decade that are involved in the control of tissue- and developmental-specific gene expression. One class of such elements, the E boxes, are interesting because they seem to be involved in the control of transcription of a number of genes, expressed in...

David Baltimore

Papers

The minimum transforming region of v-abl is the segment encoding protein-tyrosine kinase.

The effect of HeLa cell cytoplasm on the rate of sedimentation of RNA

32 The Helix-Loop-Helix Motif: Structure and Function

Direction of Polymerization by the Avian Myeloblastosis Virus Deoxyribonucleic Acid Polymerase

Purification and properties of a HeLa cell enzyme able to remove the 5'-terminal protein from poliovirus RNA.