Publisher Summary This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

Sequencing end-labeled DNA with base-specific chemical cleavages.

https://www.cell.com/cell/pdf/0092-8674(93)90530-4.pdf

Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans

3.7. Palladium Nanoparticles as Catalysts 888 3.8. Other Transition-Metal Complexes 888 3.9. Transition-Metal-Free Reactions 889 4. Applications 889 4.1. Alkynylation of Arenes 889 4.2. Alkynylation of Heterocycles 891 4.3. Synthesis of Enynes and Enediynes 894 4.4. Synthesis of Ynones 896 4.5. Synthesis of Carbocyclic Systems 897 4.6. Synthesis of Heterocyclic Systems 898 4.7. Synthesis of Natural Products 903 4.8. Synthesis of Electronic and Electrooptical Molecules 906

https://www.chem.ccu.edu.tw/~joyce/referance/ericyang/Chem.%20Rev/Chem.%20Rev.%202007,%20107,%20874-922.pdf

The Sonogashira Reaction: A Booming Methodology in Synthetic Organic Chemistry†

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.

Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics

Publisher Summary This chapter reviews the current state of the knowledge of transcription and translation in Caenorhabditis elegans. Transcription in eukaryotes is accomplished by three RNA polymerases, each transcribing a particular type of RNA; RNA polymerase I transcribes ribosomal RNA, RNA polymerase II transcribes mRNAs and snRNAs, and RNA polymerase III transcribes tRNAs and other low-molecular-weight RNAs. The chapter focuses on RNA polymerase II transcription. The C. elegans gene for the large subunit of RNA polymerase II has been cloned, and has shown to encode a protein that resembles its vertebrate counterparts. For most polymerase II transcribed genes in C. elegans, a sequence fitting the consensus TATA element about 30 bp upstream of the transcriptional start site is found. Recently, a cDNA encoding the TATA box binding protein, or TATA binding protein (TBP) subunit of transcription factor II D (TFIID), has been identified in the nematode. This protein shows extended regions of sequence similarity to vertebrate TBP, binds to a TATA box sequence and can substitute for vertebrate TFIID basal activity in in vitro extracts. These results illustrate a high degree of evolutionary conservation in the basic transcription machinery.

Transcription and translation.

Aus einer Diskussion uber den Abbau von Zuckerphosphaten wird gefolgert, das die durch Alkali bewirkte schnelle Zersetzung von Zuckerphosphaten mit freier reduzierender Gruppe in bestimmten Fallen auf Eliminierungs reaktionen und nicht auf einfache Hydrolyse zuruckzufuhren ist. – So wird Glucose-3-phosphat (I), fur das eine bequeme Synthese beschrieben wird, durch Alkali zu Glucometasaccharinsaure (IV) umgesetzt. In analoger Weise wird Ribose-3-phosphat in eine C5-Saccharinsaure umgewandelt.

Der Abbau von Glucose-3-Phosphat Durch Alkali

The effects of the acyclic analogues 3 and 4 on recognition by commonly used enzymes in recombinant DNA work have been investigated. This report shows that acyclic sugars can function in place of the normal 2′- deoxyribose moiety with some enzymes, expanding the range of possible manipulations.

Enzymatic recognition of acyclic universal base analogues in oligonucleotides

In this paper I wish to make three points. The first has to do with the chemistry of the phosphodiester linkage in phosphatidylinositols and, indeed, in phospholipids in general. The second briefly considers the interaction of the monoester phosphate residues in triphosphoinositides, and the third makes some comments on phosphotriester chemistry. A major interest of mine has been the chemistry of diesters of phosphoric acid; particularly, how neighboring groups can modify their behavior on hydrolysis. The neighboring hydroxyl group is one that frequently appears in phospholipid chemistry, and a detailed understanding of its effect was necessary before any rational structural studies could be made on the phosphoinositides. It would be generally true to say that a structure postulated for a new compound can be acceptable if the chemistry does not clash with relevant model systems. The base catalyzed hydrolysis of the glycerol inositol phosphate (FIGURE 1 ) derived from the phosphatidyl inositol from many sources conforms, in this sense, yielding two isomers each of glycerol phosphate and of inositol phosphate.1-2*3 The reasons why the product ratio is as observed have been discussed at length in terms of structural effects on ease of displacement of the leaving ester group and the ease of attack by the neighboring hydroxyl group in the formation of the intermediate cyclic phosphate^.^^^ It i s not so long ago that statements about rates of hydrolysis of phosphates could only be made in qualitative terms. Now much quantitative data are a ~ a i l a b l e . ~ ~ ~ A general observation from more recent model studies* is that in base hydrolyses involving a neighboring hydroxyl group displacement reaction, there is a direct, linear correlation between the pK, of the displaced ester group or, in other words, the stability of the corresponding alkoxide and the log of the bimolecular rate constant over a wide range. Some examples are shown in FIGURE 2 in which a propane-1,2-diol residue is used for simplicity in place of glycerol. Thus, in a general sense the relative stabilities of deacylated phosphatidyl choline, ethanolamine, inositols, and glycerols can be understood. For example, diglycerol phosphate undergoes rapid hydrolysis in base because (1) glycerol is a good leaving group as a result of its comparative acidity and ( 2 ) the 2-hydroxyl group is reactive for the same reason in attacking phosphorus. One further point may be made as a result of model work. There is an alternative degradative route that can compete with the well-recognized cyclic phosphate pathway (FIGURE 3 ) . This involves epoxide formation with displacement of a phosphate monoester group and is now well documentedP I mention this because it explains a puzzling feature of the chemistry of the phosphatidyl inositol polymannosides. The pentamannoside, for example, is more stable to alkali than the nonglycosidated compounds, but the products of hydrolysis are still glycerol phosphate and the corresponding inositol phosphate.

Aspects of phosphate ester chemistry as applied to the phosphoinositides.

The synthesis of
9-methylpyrrolo[4,3,2-de]pyrimido
[4,5-c]dihydrooxazepine 34, a
tricyclic
pyrrolo[2,3-d]pyrimidine
analogue of the mutagenic purine
N6-hydroxyadenine, and several novel
pyrrolo[2,3-d]pyrimidines is
described. The presence of the third ring constrains the amino
substituent of 34 to an anti orientation and is
expected to improve dramatically the base-pairing characteristics of the
analogue with both cytosine and thymine when present in DNA. An
intramolecular cyclisation reaction of
5-(aminooxyethyl)-4-chloro-7-methyl-2-methylsulfonyl-7H
-pyrrolo[2,3-d]pyrimidine
30 gave 33, which was converted into the target molecule 34 via
the displacement of the methylsulfonyl group with hydrazine
followed by oxidation of the hydrazino group with mercuric oxide. An
analogous cyclisation with
5-(aminooxyethyl)-4-chloro-7-methyl-7H-pyrrolo
[2,3-d]pyrimidine 31 was less
effective, whilst the corresponding 2-amino derivative 32 failed to
cyclise.

The synthesis of a tricyclic pyrrolopyrimidine related toN 6-hydroxyadenine

N(4)-Aminocytosine [N(4)NH2C] and N(4)-amino-2′-deoxycytidine [N(4)NH2dC] are highly mutagenic for Escherichia coli and phage φ 80 but not for T4. There is some evidence that they are incorporated into the φ 80 DNA but [ 14 C ]-N(4) NH 2 C could not be detected in the bacterial DNA. N(4)-Hydroxy-5,6-dihydro-6-hydroxylaminodeoxycytidine (di-NHOH-dC) is mutagenic for φ 80 and E. coli, but N(4)-hydroxydeoxycytidine [N(4)OH-dC] only has a strong inactivating effect.

Daniel M. Brown

Papers

Der Abbau von Glucose-3-Phosphat Durch Alkali

Enzymatic recognition of acyclic universal base analogues in oligonucleotides

Aspects of phosphate ester chemistry as applied to the phosphoinositides.

The synthesis of a tricyclic pyrrolopyrimidine related toN 6-hydroxyadenine

N(4)-amino- and N(4)-hydroxycytosines as base analogue mutagens