Combination of chemical and enzymatic RNA synthesis.

Identification of the Proximal Erythroid Promoter Region of the Mouse Anion Exchanger Gene

Although the field of RNA biology can trace its origin to the early 20th century, it first achieved prominence in the decade from 1955 to 1965 with pivotal breakthroughs that included the discovery of ribosomes, tRNA, and mRNA and the cracking of the genetic code. The next 30 years witnessed many other seminal discoveries, such as mRNA splicing, catalytic RNA, and regulatory RNA. To meet the needs of this flourishing field of investigation, those four decades concluded 20 years ago with the founding of the RNA Society and its monthly journal RNA. Progress in the field since then is well illustrated by advances in any number of subdisciplines. Consider the two principal fates of mRNA: translation and degradation. By 1990, much was understood about translation in bacterial and eukaryotic cells, despite the dearth of structural detail at that time. In contrast, descriptions of mRNA degradation were still largely phenomenological, even in so well studied an organism as Escherichia coli. By then, it had been established that the lifetimes of bacterial mRNAs, and even of distinct segments within the same transcript, can differ significantly from one another and that such differences in longevity can have a profound effect on gene expression. However, the basis for these differences remained a mystery, as emerging evidence that bacterial mRNA lifetimes might be governed by as yet uncharacterized features of the 5′ untranslated region was difficult to reconcile with the apparent absence of 5′ exoribonuclease activity in E. coli, whose assortment of ribonucleases is limited to endonucleases and 3′ exonucleases. Because those 3′ exonucleases were known to be impeded upon encountering base-paired regions, such as the stem–loop structures typically present at the 3′ end of bacterial mRNAs, it was hypothesized that RNA degradation in bacterial cells generally begins with internal cleavage by a low-specificity endonuclease to generate a 5′ fragment susceptible to attack by 3′ exonucleases, such as polynucleotide phosphorylase (PNPase) and RNase II, and a 3′ fragment presumed to undergo further endonucleolytic fragmentation followed by exonucleolytic digestion. However, this rudimentary mechanism provided no clue as to the identity of the endonuclease, nor did it explain how base-paired regions ever get degraded or why the 3′ product of the initial endonucleolytic event is typically shorter lived (i.e., more susceptible to cleavage) than its intact precursor. The early 1990s witnessed some key discoveries, including the identification of RNase E, which targets single-stranded regions, as the endonuclease most important for mRNA degradation in E. coli and the observation that it forms a multisubunit complex (the RNA degradosome) with PNPase and other proteins. Concurrent studies showed that base-pairing at the 5′ terminus can prolong the lifetime of RNA in E. coli by impeding internal cleavage by RNase E, a counterintuitive finding that corroborated the influence of the 5′ untranslated region on transcript stability without suggesting a mechanism. In addition, poly(A) polymerase, an enzyme activity long known to exist in bacteria but largely ignored, perhaps because of the ephemeral nature of bacterial poly(A) tails, was found to destabilize certain RNA decay intermediates by an unknown mechanism. This was roughly the state of knowledge about bacterial mRNA degradation when the first issue of RNA was published 20 years ago—a lot of phenomenology but little real understanding. What followed was a profusion of mechanistic insights, beginning with revelations about degradative events at the 3′ end. Among the first was the discovery that poly(A)polymerase facilitates the degradation of decay intermediates that end with a stem–loop by adding a 3′-terminal single-stranded tail that PNPase and the RNA helicase RhlB (another degradosome component) can use as a beachhead for launching an assault on the stem–loop preceding it. At last, a mechanism for degrading base-paired regions of RNA was in hand. Interestingly, due to the reversibility of RNA phosphorolysis, PNPase was found not only to degrade RNA in bacteria but also to add heteropolymeric tails that are A-rich, enabling this enzyme to single-handedly create unstructured 3′ ends on which it can reverse course and launch an exonucleolytic attack. In contrast, the hydrolytic 3′ exonuclease RNase II, which does not associate with an E. coli helicase, proved unexpectedly to have a protective rather than a destructive effect on base-paired 3′ ends due to its propensity to selectively remove poly(A) tails without damaging the 3′ stem–loop. Furthermore, the discovery of tmRNA-mediated trans-translation explained what happens when 3′ exonucleases advancing toward the 5′ end encounter translating ribosomes moving in the opposite direction. By releasing ribosomes trapped at the 3′ end of mRNA decay intermediates that lack a stop codon, tmRNA was found to unmask those ends and render them susceptible to 3′-exonucleolytic attack. Important progress toward understanding the role of 5′ ends in bacterial mRNA degradation soon followed. A key insight came from the surprising discovery in 1998 that the rate at which RNase E cleaves RNA at internal sites is greatly influenced by the number of phosphates at the 5′ end, monophosphorylated substrates being cut as much as 10–100 times faster than their triphosphorylated equivalents. This unprecedented property, which was later shown to be a consequence of a discrete 5′-monophosphate-binding pocket on the surface of this endonuclease, helped to explain the very short lifetime of most 3′-terminal decay intermediates, which generally bear a single phosphate at the 5′ end. However, this property alone was insufficient to account for the influence of 5′-terminal base-pairing on the longevity of primary transcripts, whose triphosphorylated 5′ ends cannot interact with RNase E. The explanation for the latter phenomenon required the subsequent discovery of the RNA pyrophosphohydrolase RppH, which converts the triphosphorylated 5′ ends of primary transcripts to monophosphorylated termini that can interact productively with RNase E if single-stranded, thereby triggering the degradation of many RNAs by a 5′-end-dependent mechanism. Meanwhile, the advent of whole genome sequencing in the late 1990s revealed major disparities in the ribonucleolytic arsenals present in different bacterial species, making it clear that E. coli could not be considered a universal paradigm for bacterial mRNA degradation. For example, despite its critical role in E. coli, where it is essential for cell growth, RNase E proved to be absent from many other bacterial species, including Bacillus subtilis and a number of important human pathogens. Instead, those organisms were found to contain alternative ribonucleases not present in E. coli, such as RNase Y (a low-specificity endonuclease) and RNase J. The subsequent discovery that RNase J is a 5′-monophosphate-dependent 5′ exonuclease explained the rapid degradation of monophosphorylated decay intermediates in species that lack RNase E as well as the polar influence of stalled ribosomes, which had previously been shown to selectively stabilize the RNA segment downstream from the stall site in B. subtilis. In recent years, considerable progress has also been made toward understanding the influence of ancillary factors on bacterial mRNA degradation, most notably the effect of noncoding RNAs that base-pair with mRNA. Though first discovered in the 1980s, the widespread influence of partially complementary small RNAs (sRNAs) and fully complementary antisense RNAs was not appreciated until many more of them were identified, beginning in the mid-1990s. It soon became clear that noncoding RNAs can either down-regulate or up-regulate gene expression, frequently by controlling the accessibility of ribosome binding sites in the mRNAs to which they bind but also by altering rates of mRNA decay. Often the influence on mRNA decay is a secondary consequence of the effect on translation, due to diminished or increased protection of the mRNA by ribosomes. However, sometimes it appears to be a direct effect of the ability of a bound regulatory RNA to create or mask an internal cleavage site or to recruit RNase E, either because the sRNA is monophosphorylated or because it is associated with Hfq, an Sm-like protein chaperone that can bind to RNase E. In the meantime, parallel studies of mRNA decay in eukaryotic cells have identified a variety of degradative pathways triggered by poly(A) tail removal, decapping, or endonucleolytic cleavage. Some of these eukaryotic pathways and the enzymes on which they rely have counterparts in bacteria, suggesting the divergent evolution of ancient RNA degradation mechanisms that date back to a common progenitor. As much as has been learned about mRNA degradation in the last 20 years, even more is not yet understood, and the next 20 years of progress in elucidating the mechanisms and biological impact of this critical regulatory process in each of the three domains of life promise to be fascinating. More generally, what do the coming decades have in store for the field of RNA biology as a whole? The essence of hypothesis-driven scientific investigation is to resolve known unknowns, which by their nature are definable if countless. As for unknown unknowns, there surely are many of those as well, RNA biology having repeatedly been reinvigorated by unanticipated discoveries that have enabled the field to reinvent itself. Find a seat. The sequel is about to begin.

TFIIF, ELL, and Elongin belong to a class of RNA polymerase II transcription factors that function similarly to activate the rate of elongation by suppressing transient pausing by polymerase at many sites along DNA templates. SII is a functionally distinct RNA polymerase II elongation factor that promotes elongation by reactivating arrested polymerase. Studies of the mechanism of SII action have shown (i) that arrest of RNA polymerase II results from irreversible displacement of the 3'-end of the nascent transcript from the polymerase catalytic site and (ii) that SII reactivates arrested polymerase by inducing endonucleolytic cleavage of the nascent transcript by the polymerase catalytic site thereby creating a new transcript 3'-end that is properly aligned with the catalytic site and can be extended. SII also induces nascent transcript cleavage by paused but non-arrested RNA polymerase II elongation intermediates, leading to the proposal that pausing may result from reversible displacement of the 3'-end of nascent transcripts from the polymerase catalytic site. On the basis of evidence consistent with the model that TFIIF, ELL, and Elongin suppress pausing by preventing displacement of the 3'-end of the nascent transcript from the polymerase catalytic site, we investigated the possibility of cross-talk between SII and transcription factors TFIIF, ELL, and Elongin. These studies led to the discovery that TFIIF, ELL, and Elongin are all capable of inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates. Here we present these findings, which bring to light a novel activity associated with TFIIF, ELL, and Elongin and suggest that these transcription factors may expedite elongation not only by increasing the forward rate of nucleotide addition by RNA polymerase II, but also by inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates.

The basic polymerase chain reaction (PCR) (1), which consists of the exponential amplification of DNA fragments between terminal sequences recognized by specific oligonucleotide primers, is a very powerful method but does not satisfy all wishes. The need for sequence information from the DNA template, in order to create the primers, limits the applicability of the method to a few organisms or to highly conserved genomic DNA regions. This is a handicap if highly variable regions need to be analyzed, as is normally the case for the identification of organisms at the strain or isolate level. To circumvent this problem, the basic PCR method has been modified. Instead of long oligonucleotide (20–30 mer) primers, short (9–13 mer) oligonucleotides of arbitrary sequence can be used. They bind to complementary sequences occurring more or less frequently in the genome. A DNA amplification product is generated for each genomic region that happens to be flanked by a pair of priming sites that are within 3000 bp of each other and in the appropriate orientation (Fig. 1). Amplification of genomic DNA with short arbitrary primers. Fragments, which are flanked by a pan of priming sites within 3000 basepans in the appropriate orientatton (A), are amplified exponentially (A′). Fragments with one priming site (B) or with more than 3000 bp within a pan of priming sites (C) are not amplified exponentially (B′, C′ ). KeywordsArbuscular MycorrhizalSingle SporeEthidium Bromide SolutionDilute Stock SolutionSpore PreparationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The Role of Deoxyribonucleic Acid in Ribonucleic Acid Synthesis: X. THE INFLUENCE OF 6-METHYLAMINOPURINE ON NUCLEIC ACID SYNTHESIS IN VITRO

NusA protein is a transcription elongation and termination factor that acts to enhance pausing of RNA chain growth by RNA polymerase at specific sites on DNA templates. We demonstrate that this enhancement of pausing in tR1, the transcription termination site between genes cro and cII of phage lambda, is inhibited by DNA oligonucleotides complementary to a segment of the nascent RNA just preceding the sequence that is thought to be a part of the stem of an RNA hairpin that is responsible for pausing, but not by oligonucleotides complementary to segments containing the boxA sequence or the sequences up to 40 nucleotides upstream from boxA. The results are consistent with a model proposed by Landick and Yanofsky (Landick, R., and Yanofsky, C. (1987) J. Mol. Biol. 196, 363-377) in which the NusA protein acting as a subunit of RNA polymerase in the elongation complex contacts a structure near the 3' end of the nascent transcript and uses the binding energy to shift the equilibrium between elongation and paused conformational isomers of the transcription complex in favor of the paused isomer.

A series of adenosine triphosphate (ATP) derivatives bearing chloro, fluoro, amino, methyl, vinyl, and ethynyl groups at position 2 are synthesized and tested as substrates for RNA and DNA polymerases. The modified nucleotides work well in in vitro transcription with T7 RNA polymerase and primer extension (PEX) using engineered DNA polymerases (TGK, 2M) except for the bulkier 2‐vinyl‐ and 2‐ethynyl‐ATP derivatives that give truncated products. However, in single nucleotide incorporation followed by PEX, they still can be used for site‐specific incorporation of reactive modifications into RNA that can be further used for postsynthetic labeling through thiol‐ene or Cu‐catalyzed alkyne‐azide cycloadditions reactions. All modified ATPs work in polyadenylation catalyzed by poly(A) polymerase to form long 3′‐polyA tails containing the modifications that also can be used for labeling.

RNA/DNA hybrids in human immunodeficiency virus (HIV) replication are cleaved by HIV-1 reverse transcriptase (RT) H in locations determined by hybrid structure. Minus strand DNA synthesis is accompanied by cleavage of template viral RNA directed by RT positioned at the growing 3' DNA end. Some RNA remains as oligomers annealed to the new DNA strand and is cut by RTs positioned at the 5' RNA ends. We constructed substrates to the test the hypothesis that internal helix structure, rather than strand end structure, drives the RT to position at 3' DNA and 5' RNA ends. On substrates with an RNA primer recessed on a DNA template, the 5' end of the RNA had a dominant role in the determination of RNase H cleavage positions. If the 5' end region of the RNA could not anneal, cleavage would not occur. Nevertheless, we obtained evidence that helix structure promotes the binding of RT to the end of the helical region closest to the 5' RNA/3' DNA end. When a DNA primer recessed on an RNA template had a 3' unannealed region, cleavage occurred, with RT positioned solely by helical structure at the 5' RNA/3' DNA end of the annealed region of the hybrid. Using substrates having RNA primers annealed to circular DNA templates, we showed that cleavage can be independent of the presence of a DNA 3'end and is directed by the 5' RNA end. Overall, the results suggest that the RT initially binds an internal region of the hybrid and then is driven in the direction to encounter a 3' DNA or 5' RNA end, where it is positioned for catalysts by the strand end. The requirement for two modes of RNA cleavage in viral replication and the unexpected requirement for the 5' RNA end structure are discussed.

Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are essential nucleic acid building blocks and serve as energy molecules for a wide range of cellular reactions. Cellular GTP concentration fluctuates independently of ATP and is significantly elevated in numerous cancers, contributing to malignancy. Quantitative measurement of ATP and GTP has become increasingly important to elucidate how concentration changes regulate cell function. Liquid chromatography–coupled mass spectrometry (LC–MS) and capillary electrophoresis-coupled MS (CE–MS) are powerful methods widely used for the identification and quantification of biological metabolites. However, these methods have limitations related to specialized instrumentation and expertise, low throughput, and high costs. Here, we introduce a novel quantitative method for GTP concentration monitoring (GTP-quenching resonance energy transfer (QRET)) in homogenous cellular extracts. CE–MS analysis along with pharmacological control of cellular GTP levels shows that GTP-QRET possesses high dynamic range and accuracy. Furthermore, we combined GTP-QRET with luciferase-based ATP detection, leading to a new technology, termed QT-LucGTP&ATP, enabling high-throughput compatible dual monitoring of cellular GTP and ATP in a homogenous fashion. Collectively, GTP-QRET and QT-LucGTP&ATP offer a unique, high-throughput opportunity to explore cellular energy metabolism, serving as a powerful platform for the development of novel therapeutics and extending its usability across a range of disciplines.Graphical

This month's issue of the Archives of Pathology & Laboratory Medicine includes a unique special section on "Molecular Signatures of Lung and Pleural Tumors" that represents the proceedings of a special Joint Symposium of the European Working Groups for Molecular Pathology and Pulmonary Pathology at the 21st European Congress of Pathology in Istanbul, Turkey, in September 2007. This symposium and the subsequent special section were organized by Dr Helmut Popper of the Institute of Pathology at the Medical University of Graz, Graz, Austria, president elect of the Austrian Society of Pathologists and immediate past president of the European Working Group for Pulmonary Pathology. In this brief review, we have provided some definitions of terms and concepts used in the proceedings of the "Molecular Signatures of Lung and Pleural Tumors" special section for those readers who are not already familiar with molecular pathology.Molecular pathology may have once been a specialized component of the research laboratory or the clinical laboratory, but today molecular diagnostic and prognostic techniques are in common use within the anatomic pathology laboratory, especially in the realm of infectious organism diagnosis and cancer diagnosis. Molecular testing continues to expand as more easily obtainable archival paraffin-embedded tissue replaces fresh and frozen tissues as the source of DNA and RNA needed for molecular analysis, and as newer technologies allow for more streamlined methods of testing. This review addresses the increased application of molecular testing in lung pathology, specifically how the current state of molecular pathology may be applied to practical, everyday lung pathology diagnosis.Genes, made up of nucleic acids, contain the information necessary for the construction of proteins from amino acids within a cell. Genes code for proteins required for metabolic reactions and cellular structure. DNA makes up genes, and RNA transcribes the genetic code held within the DNA into proteins. The genetic code within the genes is composed of nucleic acids, for which nucleotides are the building blocks. Nucleotides, made up of a sugar-phosphate backbone with a nitrogenous base, are either purines—adenine (A) and guanine (G) in DNA and RNA— or pyrimidines—thymine (T) and cytosine (C) in DNA (uracil [U] replaces T in RNA). The nucleotides that make up the genes are arranged in a double-stranded right-handed helix. Nucleotides in DNA are arranged sequentially so that a gene will code for a matching protein. Within a double helix pattern, A, a purine, always binds with T, a pyrimidine, and G always binds with C, giving a nucleotide sequence for which 1 strand is a "mirror image" of the other strand.1–6There are 46 chromosomes (23 pairs) in a human diploid cell, on which all genes are located. Chromosomes are paired, and as such a gene is found on a locus on each of the 2 paired chromosomes, giving 2 copies, or alleles, of genes. Gametes are haploid rather than diploid and therefore contain only 1 allele for each gene. Diploid status is reestablished when the nuclear material from an egg and sperm combine during fertilization.1–6Transcription, the synthesis of messenger RNA (mRNA) from a DNA strand, is a key step in the formation of protein coded by DNA. During transcription, enzymes called topoisomerases break a DNA strand and allow the DNA double-helix to uncoil, giving 2 DNA strands, 1 of which is the template for mRNA, called the DNA template. Base pairs are matched with the DNA template to produce a mirror image of the DNA template, except with the substitution of U for T, forming a strand of mRNA. A series of 3 base pairs in a gene, called a codon, code for a specific amino acid, so that a series of codons code for a particular sequence of amino acids resulting in the synthesis of a specific protein. Translation, the assembly of the protein molecule from the mRNA template, occurs with the addition of amino acids in a particular sequence based on the specificity of the mRNA. Transfer RNA assists in translation.1–6After translation, modifications to the newly formed protein occur in order for it to function, to move within the cell, or to fold properly. Methylation, acetylation, phosphorylation, glycosylation, posttranslational cleavage, and the addition of lipid groups are examples of posttranslational modifications. End regions of chromosomes are made up of telomeres, hundreds of repeats of the nucleotide sequence TTAGGG. Some of these telomere sequences are lost each time a cell divides, until they are lost and the cell can no longer divide. This process is called senescence. A polymerase called telomerase is able to replace the DNA sequences at the end regions, allowing for continuing cell division—a significant feature in some cancers.1–6The polypeptide chain formed by the specific amino acid sequence causes the newly formed protein to fold into a tertiary arrangement giving it a 3-dimensional structure. Often, the newly formed protein is inert until made functional by a posttranslational modification such as phosphorylation or proteolytic cleavage. Phosphorylation, the addition to the protein of a phosphate group catalyzed by enzymes called kinases, may cause, for example, translocation of the protein from the cytosol into the nucleus. Dephosphorylation is the removal of a phosphate group catalyzed by enzymes called phosphatases. Phosphorylation and dephosphorylation of proteins are often important in the activation and deactivation of cell cycle proteins, signaling pathway proteins, and transcription factor proteins.1–6Protein degradation is necessary to remove damaged proteins and limit signaling proteins such as those involved in cell survival and cell death. This degradation often needs to proceed quickly. Reversible cross-linkage to a polypeptide, termed ubiquitin, leads to the rapid degradation of proteins and is called ubiquinylation or polyubiquinylation.7The control of gene expression is for the most part controlled by regulation of transcription initiation. Proteins called transcription factors, also termed transactivators or trans-acting factors, bind to DNA and regulate RNA polymerase activity, affecting gene expression either by inducing or activating the gene or by inhibiting the gene by reducing transcription levels.8–13 Transcription factors are necessary for RNA polymerase to initiate transcription, and transcription factors called transcriptional activators stimulate transcription of an RNA molecule from its DNA template.1415A variety of techniques exist for molecular pathology diagnosis, including nucleic acid extraction, Southern blotting, restriction fragment length polymorphism, sequencing, liquid bead microarrays, mass spectrometry, and comparative genomic hybridization, among others. Nucleic acid extraction historically has used organic techniques using chloroform and phenol; however, automated nucleic acid extraction exists today and is frequently used to purify nucleic acids for their use with other molecular methods. For practical laboratory-based molecular diagnosis of lung disease, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are 2 of the most important techniques commonly used.In situ hybridization uses DNA or RNA probes to evaluate intact cells for genetic changes. Probes visualized with a chromogen that produces a colored chemical at the reaction site is called chromogenic in situ hybridization and probes using fluorescent labels are called FISH. Evaluation of genetic alterations within intact cells allows for the detection of genetic alterations occurring in a specific group of cells or within a small number of examined cells and is a major benefit of the use of in situ hybridization in anatomic pathology. It is commonly used with cytologic and surgical specimens to detect tumor cells and certain microorganisms, and with surgical specimens of tumors for its prognostic utility and to determine treatment response.16–22 Chromogenic in situ hybridization uses a probe that can be seen as a chromogenic reaction under light microscopy, whereas FISH is available as probe sets and multiprobe FISH cocktails. Peripheral blood, urine, sputum, endoscopic brushings and washings, and paraffin-embedded, formalin-fixed tissue are all suitable for FISH. However, fixation of tissue with formalin for longer than 48 hours may yield poorer FISH results.23DNA and RNA probes hybridize to a specific target sequence that is of interest, for example, genes implicated in a specific type of cancer or an inherited disease, or to a certain microorganism.16–22 Probes are made using DNA fragments cloned from yeast or bacterial artificial chromosomes and, with FISH, are directly or indirectly fluorophore labeled, most often using Texas Red, fluorescing red, and fluorescein isothiocyanate, fluorescing green.24 Directly labeled probes have a fluorophore-labeled nucleotide inserted into the probe, so binding of the probe to its target in 1 hybridization step is all that is required to visualize the probe. Indirectly labeled probes have a reporter molecule such as biotin or digoxigenin attached covalently, requiring the additional step of the application of a fluorophore-labeled avidin or fluorophore-labeled antidigoxigenin. The additional step required with indirectly labeled probes is a disadvantage; however, indirectly labeled probes generally allow for stronger signals because of greater signal amplification.24 There are 4 general types of probes: chromosome enumeration probes, locus-specific indicator probes, telomeric probes, and chromosome paints. Chromosome enumeration probes hybridize to repetitive DNA sequences located near chromosome centromeres, and because the loss of a centromere is generally indicative of the loss of an entire chromosome, they are used to enumerate the number of copies of a certain chromosome within a cell.2526 Locus-specific probes are probes to unique sequences and are most frequently used to determine whether specific genes are amplified, translocated, or deleted. Telomeric probes hybridize to unique DNA sequences located very close to telomeres. The probes do not hybridize to telomeric sequences. Chromosomal paints are a mix of probes that probe to the entire length of 1 or more chromosomes.A FISH specimen must undergo prehybridization to allow a probe to efficiently hybridize to cellular DNA targeted by the probe while protecting the cell from morphologic disruption. Following prehybridization, the probe and cellular DNA are denatured so that the probe can hybridize to the cellular DNA it is targeting. Hybridization usually takes between 4 and 12 hours. A type of DNA called Cot DNA is added during hybridization to hybridize highly repetitive DNA sequences that are located within the genome so that the probe DNA does not nonspecifically bind these repetitive sequences and yield a multitude of nonspecific signals rather than the appropriately specific signal.24252728 The nonbound probe is then removed by washing, a nuclear counterstain that weakly fluoresces is added so that the nucleus can be identified, and an antifade is added to retard fluorophore photobleaching. The signal produced by the FISH fluorophore is then examined with fluorescence microscopy.Since its introduction in 1985,29 PCR has been refined to be an efficient and sensitive method of studying the molecular pathology of primary and metastatic neoplasms, inflammatory mechanisms, and infectious diseases.30–37 Today, automated instruments designed for the laboratory tabletop are available. Polymerase chain reaction amplifies DNA via a repeated 3-step process of denaturation, annealing, and extension. Double-stranded DNA that is the target is denatured at high temperature to yield 2 intact single strands of complementary DNA. Then at a lower temperature, specially designed single-stranded DNA primers anneal or bind to a specific targeted area of the single-stranded DNA. Because there are very large numbers of DNA primers relative to the full-length complementary DNA strand, the target DNA anneals with the primer DNA much more frequently than with the complementary DNA when cooling occurs. In the third step, extension, Taq polymerase identifies the now partially double-stranded DNA and extends the primers by polymerization, to yield, at the end of the first cycle, 2 double-stranded copies of a portion of the target DNA generated from 1 copy. Because the DNA primers only recognize the DNA for which they have been specifically designed, only that specific segment of DNA, making up a small part of the entire DNA present in the original DNA, is preferentially amplified. Subsequent cycles produce numerous shorter double-stranded DNA PCR products, so that more than a billion copies of the original double-stranded DNA are produced after 30 cycles, and more than a trillion are produced after 40 cycles. Following amplification, post-PCR analysis of the markedly increased amount of targeted DNA sequence product can be performed. The target sequence can be shown to be present in a specimen by the use of amplicons run on polyacrylamide or agarose electrophoresis, or via Southern blotting with probe hybridization, comparing the lengths of the targeted DNA sequence with DNA "ladder" markers. DNA sequencing can be performed on the amplicons, or studies to identify mutations may also be performed.3839 Because PCR enormously amplifies DNA, great caution must be used in performing PCR to avoid cross-contamination of a specimen with even very small amounts of DNA.Real-time PCR is becoming a more and more popular method of molecular pathology research and diagnosis that eliminates the need for post-PCR analysis and allows for relatively quick detection of DNA targets, including specific mutations.40–44The best way to examine specific genes present in a certain cell type, such as in tumor cells, is to examine those cells' mRNA. As RNA is not stable enough to work with easily in a laboratory, reverse transcription can be used to convert mRNA into its complementary DNA. With reverse transcription, mRNA is the template used for the production of a strand of DNA, opposite or reverse of typical cellular transcription. The complementary DNA can be used as the template for PCR in a process called reverse transcriptase–PCR (RT-PCR). Reverse transcriptase–PCR can be used to examine genes that are expressed, overexpressed, underexpressed, or not expressed in a specific cell type by the isolation of specific mRNA.44–47 Altered gene expression is a characteristic of malignant transformation, and those alterations allow for the identification of the presence of cancer cells via the detection of mRNA transcripts specific to those tumor cells. Tumor markers have been identified that are specific to solid organ cancers, and RT-PCR is highly sensitive in detecting differentially expressed tumor-related mRNAs. Some studies have indicated that RT-PCR can detect as few as 1 cancer cell in a million normal cells.4448 Real-time quantitative RT-PCR has become popular for detecting and quantifying RNA targets in a variety of cancers. It requires no post-PCR analysis and is efficient and automated. Several studies have used real-time quantitative RT-PCR to evaluate lymph nodes for micrometastases, including for non–small cell lung carcinoma (NSCLC), and to examine peripheral blood for potential dissemination of lung cancer cells during lobectomy.49–52Normal human DNA contains 2 alleles for every genetic locus, the majority of which are identical, or homozygous, and the loss of 1 allele results in no pathologic change. Some genetic loci have 2 differing copies of alleles and are heterozygous. The majority of these heterozygous alleles allow for normal variance and do not result in pathologic changes; however, some of these heterozygous loci have the potential to cause pathologic genetic variations, with resultant disease. If there is a loss of the normal, or "wild-type," allele at a locus, with resultant "loss of heterozygosity," the remaining aberrant allele can cause cellular damage. At homozygous loci, the loss of an allele can occur and be followed by gene silencing or point mutation, causing loss of tumor suppressor genes.5354 Detection of loss of heterozygosity using older methods such as Southern blot analysis and restriction fragment length polymorphism analysis are low-throughput, tedious, and inefficient; however, newer, high-throughput single nucleotide polymorphism arrays have allowed for more efficient examination of loss of heterozygosity.55–57Cancers, including lung cancers, commonly exhibit loss of heterozygosity, causing the inactivation or silencing of genes critical for growth regulation and homeostasis. Cigarette smoking has been associated with loss of heterogeneity of sites on chromosome 3, and the association is greater in patients who began smoking at a young age.58–61 More than 90% of small cell carcinomas and more than 70% of NSCLCs contain loss of heterozygosity.576263 Among NSCLCs, squamous cell carcinomas exhibit loss of heterozygosity in more than 90% of cases, compared with adenocarcinomas, showing loss of heterozygosity in approximately 70% of cases. In NSCLC, loss of heterozygosity generally involves genetic foci on chromosomes 1p, 3p, 8p, 9p, 13q, 17p, 19p, Xp, and Xq. In small cell carcinomas, loss of heterozygosity generally involves chromosomes 3p, 4q, 5q, 4q, 10q, 13q, 15q, and 17p.5762–66 Losses found in both small cell carcinomas and NSCLCs, involving chromosomes 3p, 13q, and 17p, are probably related to inactivation of critical tumor suppressor genes including retinoblastoma, p53, and fragile histidine triad (FHIT).576263 Loss of heterozygosity in premalignant conditions and malignant diseases of the lung represents both early- and late-stage changes in the progression of disease; however, the continuum of losses makes it hard to evaluate the specific contribution of each loss. Loss of heterozygosity has also been identified in some benign lung diseases, including asthma and chronic obstructive pulmonary disease, probably reflecting the genetic predisposition identified in these diseases.67–70 Loss of heterozygosity has also been identified in cases of usual interstitial pneumonia (idiopathic pulmonary fibrosis) and suggests premalignant potential in those cases.71Extracellular messenger molecules such as hormones, inflammatory cytokines, and growth factors, called ligands, bind to specific cell surface receptors and activate messengers within the cytosol leading eventually to activation of nuclear transcription factors that, due to the extracellular message, direct the transcription of a specific gene product, such as the transcription of a protein involved in cell growth. This cascade of events is termed signal transduction, and the series of steps within the cascade is termed signal transduction pathway or signaling pathway. Growth factor receptors are a common cell surface receptor, on which polypeptide growth factors such as epidermal growth factor are ligands attaching to those receptor protein-tyrosine kinases, activating the receptor and causing it to bind with intracellular proteins, which in turn continue the signaling pathway. Epidermal growth factor receptor is a member of the type I growth factor receptor tyrosine kinase family. Epidermal growth factor receptor has other ligands that bind to it other than epidermal growth factor, including transforming growth factor α, and these ligands, receptors, and signaling pathways play a central role in many lung cancers as well as some nonneoplastic pulmonary diseases.72–77The extracellular ligands' "messages" are transmitted via a signaling pathway. Cell differentiation and proliferation, cell survival, and cell death and apoptosis are regulated by signaling pathways, the majority of which "cross-talk" with other signaling pathways in a complex manner. Several important signaling pathways have been well studied. For example, the Wnt/B/catenin pathway, termed the canonical Wnt signaling pathway, involves Wnt binding to Frizzled cell surface receptors, which in turn activate Disheveled, causing the inhibition of protein kinase glycogen synthase kinase 3, which in turn releases dephosphorylated β-catenin from the adenomatous polyposis coli–axin complex. β-Catenin associates with T-cell factor/lymphoid enhancer–binding factor transcription factors causing the induction of Myc.78–84 Other important signaling pathways include the JAK/STAT pathway, involving signal transducers and activators of transcription (STAT) proteins and Janus kinase (JAK) nonreceptor protein tyrosine kinases; the Ras/Raf-1/MAPK pathway, a significant pathway in carcinogenesis, including epithelial cell proliferation; the nuclear factor-κB transcription factor and nuclear factor-κB signaling pathways that regulate immune system proteins, cell survival and proliferation proteins, and apoptosis proteins; and the PI3K/Akt/ mTOR pathway important in regulating cell survival; among many others.85–93Errors in replication, extracellular influences such as UV light, radiation, and chemicals, and endogenous influences such as oxygen radicals routinely cause DNA damage, generally depurination, and with the of a chemical group to DNA, the of which is termed an Several DNA pathways exist to the damaged DNA and replace it with newly DNA based on its complementary DNA DNA pathways are important in an to lung cancer and to lung cancer The base pathway small foci of DNA including or single or fragments and The nucleotide pathway DNA involving both The be because no for the DNA segment is available. can cause DNA such as chemical and The DNA pathway, also termed the break pathway, involves a cascade of events and to double-stranded The direct pathway DNA by in cell cycle is a series of very regulated events cell proliferation, including into DNA replication, replication, into cell cell and cell The cell cycle is into at for DNA synthesis or of and with nuclear and cellular which control of DNA and cell in to and with proteins called that regulate the progression of the series of steps in the cell cycle by activating and proteins by phosphorylation, including proteins that as on cell cycle progression and cell The cell cycle is controlled by many pathways and and and it may be appropriately or in inflammatory Cell cycle regulation loss is a very important step in cell proliferation during cycle damaged DNA to be on to cells by the cell cycle at specific It allows damaged DNA to be the is to cells into apoptosis cell The primary in the cell cycle is the restriction point in to the cell cycle occurs. Other include an and a A complex of proteins, the complex and the detect DNA at these DNA is the DNA is and the cell cycle factor signaling the cell cycle and the the however, once the cell the cell restriction it no longer requires growth factor and the cell is to the cell The gene product, progression past the restriction point of the cell cycle and the expression of genes involved in DNA of the cell cycle also on activation of of the of p53, the gene product, is to the DNA the of the cell cycle at in to DNA or to the induction of apoptosis when is As has been to as the of the and have critical in the of the cell cycle, and of and are the most common associated with the cell cycle of however, due to the large number of and pathways, many can produce direct or loss. The gene for 2 protein products, a kinase and of either of these can produce of the or of and as well as other genes and their or of and p53, can produce loss of cell cycle control the direct loss of and and prognostic techniques using molecular are used in anatomic pathology genes for proliferation, and the used for of molecular and factors involved in genetic will the current and molecular testing and their to the diagnosis and of lung

BackgroundRNA is known to play diverse roles in gene regulation. The clues for this regulatory function of RNA are embedded in its ability to fold into intricate secondary and tertiary structure.ResultsWe report the transcriptome-wide RNA secondary structure in zebrafish at single nucleotide resolution using Parallel Analysis of RNA Structure (PARS). This study provides the secondary structure map of zebrafish coding and non-coding RNAs. The single nucleotide pairing probabilities of 54,083 distinct transcripts in the zebrafish genome were documented. We identified RNA secondary structural features embedded in functional units of zebrafish mRNAs. Translation start and stop sites were demarcated by weak structural signals. The coding regions were characterized by the three-nucleotide periodicity of secondary structure and display a codon base specific structural constrain. The splice sites of transcripts were also delineated by distinct signature signals. Relatively higher structural signals were observed at 3’ Untranslated Regions (UTRs) compared to Coding DNA Sequence (CDS) and 5’ UTRs. The 3′ ends of transcripts were also marked by unique structure signals. Secondary structural signals in long non-coding RNAs were also explored to better understand their molecular function.ConclusionsOur study presents the first PARS-enabled transcriptome-wide secondary structure map of zebrafish, which documents pairing probability of RNA at single nucleotide precision. Our findings open avenues for exploring structural features in zebrafish RNAs and their influence on gene expression.

Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3' end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli, the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3' end of a nascent transcript during transcription initiation. Immediately following the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promoter sequences. One of the operons regulated (in part) by UTP-sensitive reiterative transcription in E. coli is the carAB operon, which encodes the first enzyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucleotide sequence at and near the transcription start site and spacing between the start site and -10 region of the promoter on reiterative transcription and gene regulation. Our results indicate that these variables are important determinants in establishing the extent of reiterative transcription, levels of productive transcription, and range of gene regulation.

Nontranslatable transcripts containing interspersed repetitive sequence elements constitute a major fraction of the poly(A) RNA stored in the cytoplasm of both the sea urchin egg and the amphibian oocyte. We report the first complete sequence of a representative interspersed maternal RNA transcript, called ISp1. The transcript is about 3.7 kb in length [including poly(A) tail]; and the 5' half consists of a cluster of repetitive sequences, whereas the 3' half is single copy. Other repetitive sequences occur in the 5' and 3' regions flanking the transcription unit. In several cloned alleles, the flanking repetitive and single-copy sequences differ, indicating a high degree of insertional and deletional rearrangement around, as well as within, the transcription unit. No significant open reading frames exist in any region of the ISp1 transcript, nor is it spliced to give rise to translatable mRNA in egg or embryo. A 620-nucleotide repetitive sequence element at the 5' end of the ISp1 transcript is also represented in a large number of other long interspersed maternal poly(A) RNAs. In addition, this sequence appears in a prevalent set of small polyadenylated RNAs about 600-nucleotides in length, which disappear almost completely by the gastrula stage of development. The structural features of the ISp1 RNA uncovered in this work exclude several hypotheses of interspersed maternal poly(A) RNA origin and function.

Driven by the concern about the future petro-chemical feedstock availability, as well as the need for a green and sustainable society, renewable and bio-based materials have become the natural candidates for alternatives to petro-chemicals. As the most abundant biomass feedstock, carbohydrates are attracting significant attention from both academia and industry for the production of chemicals, fuels and energy. However, one major drawback of directly using carbohydrates in industrial processes is their limited thermal stability caused by the existence of multiple functional groups, for instance hydroxyl groups. Consequently, simplified or bi-functional carbohydrate-derived building blocks are often more attractive than the virgin carbohydrates for polymer chemists. In this thesis, we aim to 1) develop a new family of difunctional bio-based building blocks, and 2) utilize these new building blocks as AA monomers to synthesize step-growth polymers, such as polyesters and polyamides. Ideally, the new polymers are expected to be semicrystalline, preferably having high melting (Tm) and high glass transition temperatures (Tg), and can be used as the potential fully or partially bio-based engineering plastics. Isohexides (1,3:4,6-dianhydrohexitols), a group of secondary, rigid diols, as well as their derivatives are interesting carbohydrate-derived building blocks for various types of step-growth polymers, such as polyesters, polyamides and polycarbonates. Due to the intrinsic rigidity of the structures, isohexides are capable of increasing the glass transition temperatures when incorporated into polymers. However, the relatively poor reactivity of the secondary hydroxyl groups of isohexides has been recognized as a main drawback, which often results in low molecular weight or discolored polymers. In order to overcome these problems, as well as utilizing the rigidity of the isohexide skeleton, a new family of 1-carbon extended isohexide-derivatives were designed and synthesized by transforming the less reactive secondary hydroxyl functionalities into more reactive ones. Based on a key dinitrile intermediate, viz. isoididedinitrile (IIDN), the novel isohexide-derived building blocks were obtained. They are: isoidide-2,5-dicarboxylic acid (IIDCA) and its dimethyl ester (IIDMC), isoidide-2,5-dimethanol (IIDML) and isoidide-2,5-dimethyleneamine (IIDMA). After optimization of the respective synthetic routes, these building blocks were obtained at sufficient scales (5–20 g/batch) and high purities (>99.5%) to be suitable for polymerizations. Three series of polyesters based on the novel 1-carbon extended isohexides were synthesized. The first series of polyesters is based on isoidide dicarboxylic acid (IIDCA) and linear alkylene diols. Unexpectedly, they have rather low Tm (60–100 °C) values whereas the recorded low Tg values (-30–18 °C) were approximately expected. The second series of polyesters is fully based on renewable, isohexide-derived monomers. The combination of both isohexide-based AA/BB monomers gave significantly higher Tg values (45–85 °C) than the partially linear aliphatic polyesters of the first series. Importantly, this part of the project revealed the promising characteristics (reactivity, rigidity and thermal stability) of isoidide-2,5-dimethanol (IIDML) for potential applications in performance polymers with high Tg and Tm. Therefore, the third series of polyesters was entirely focused on IIDML-based semi-aromatic systems. The Tg and Tm values of these polyesters were found to exceed even those of the conventional PET and PBT polyesters, confirming our expectation that IIDML is a promising rigid cyclic diol for engineering plastic applications. Two series of semicrystalline (co)polyamides were synthesized from the new isoidide 2,5-dimethyleneamine (IIDMA) or from a mixture of IIDMA and 1,6-hexamethylene diamine with the biobased dicarboxylic acids sebacic acid or brassylic acid. A combination of melt polymerization of nylon salts with solid-state polycondensation (SSPC) processes afforded the desired copolyamides with maximum number average molecular weight (Mn) of 49,000 g/mol. Analysis by temperature dependent soldi state NMR and FT-IR spectroscopies revealed that the new diamine IIDMA exist in both the crystalline and amorphous phases of the copolyamides. Given the sufficiently high Tm values (~200 °C) of the copolyamides containing less than 50% of IIDMA, these bio-based semicrystalline copolyamides can be useful for engineering plastic applications. By comparing the thermal properties of the polymers based on the new isohexide-based building blocks, i.e. IIDCA, IIDML and IIDMA, with relevant reference polymers, systematic structure-thermal properties studies of each series of polymers were conducted. This work objectively evaluated the relative rigidities, reactivities and thermal stabilities of these new building blocks with respect to those of the parent isohexides, as well as with respect to their linear, cyclic aliphatic and aromatic analogues. Both the experimental (i.e., single crystal X-ray diffraction) and computational-aided methods (i.e., Density Functional Theory (DFT)) were employed to elucidate the structural characteristics of the isohexide-based building blocks, as well as to understand the specific thermal properties of the polymers based thereon. Additional efforts were invested to expand the dinitrile platform to the other stereo-isomers. The isomeric isohexide dinitriles were synthesized by base-induced epimerization. The strong non-nucleophilic organic base 1,8-diazabicycloundec-7-ene (DBU) was found to be a suitable catalyst for such type of isomerization. A kinetic study on the epimerization of three dinitrile isomers revealed an equilibrium between the three possible isomers in THF. Single crystal X-ray diffraction studies demonstrated several structural characters of these molecules regarding their symmetry and linearity. Density Functional Theory (DFT) calculations further supported the base-induced epimerization mechanism. This work concerns a pioneering study of deoxy-1-carbon-extended isohexide-based building blocks. All the monomers and polymers synthesized, characterized and evaluated in this thesis are entirely new. Suitable synthetic laboratory-scale protocols were developed for all the monomers and polymers. The reported study of the structure-thermal properties relations, as well as the crystal structure elucidation by single crystal X-ray diffraction and DFT, are also of high importance concerning the field of isohexide-based polymers and will certainly prove to be very useful for drawing conclusions on the industrial feasibility of such bio-based materials.

DNA-dependent synthesis of tryptophan (trp) operon mRNA and enzymes was studied in vitro. With φ80 trpEDCBA template DNA trp operon transcription was initiated primarily at a phage promoter estimated to be approximately 7400 to 8000 nucleotides from trpE. Sequential transcription of trp mRNA in the correct direction proceeded at a rate of about 19 nucleotides per s. Trp mRNA resulting from read-through transcription from the phage promoter was larger than 23 S. Coupled sequential synthesis of trp operon enzymes occurred at a rate of approximately 4 amino acids per s. Experiments were conducted to determine if ribosomes discharge from polycistronic trp mRNA following translation termination at natural chain termination codons. Kasugamycin was used to inhibit reinitiation of translation. Kasugamycin shut down of steady state translation required 6.5 min for trpE enzyme, 7 min for trpD enzyme, and 3 min for trpA enzyme. These results indicate that ribosomes which translate trpE mRNA must discharge and reattach in order to translate trpD mRNA, and that ribosomes that translate trpD mRNA must discharge and reattach in order to translate trpA mRNA. Kasugamycin inhibition of trpD enzyme and trpA enzyme synthesis under appropriate conditions of sequential transcription and coupled translation provided further evidence for obligatory discharge and reattachment of ribosomes. Enzyme synthesis coupled to trp operon-promoted transcription from λh80 trp template DNA was repressible by tryptophan in reaction mixtures containing S-30 extract from trpR+ cells. In contrast, enzyme synthesis coupled to read-through transcription from φ80 trp template DNA was essentially irrepressible. It is concluded that read-through transcription is not repressible in vitro. Trp operon mRNA transcribed from a λh80 trpEDCBA DNA template was smaller than 23 S. The polar mutation trpC6 was expressed in vivo in phage λh80 trpEDCBA 190-9 but not in phage φ80 trpEDCBA 190. Read-through transcription of the trp operon occurs in the latter phage but not in the former. Polarity was not expressed in vitro using template DNA from either phage. Trp operon mRNA was extremely stable in vitro; the half-life varied between 30 min and more than 2 hours.