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The modern version of the RNA World Hypothesis begins with activated ribonucleotides condensing (nonenzymatically) to make RNA molecules, some of which possess (perhaps slight) catalytic activity. We propose that noncanonical ribonucleotides, which would have been inevitable under prebiotic conditions, might decrease the RNA length required to have useful catalytic function by allowing short RNAs to possess a more versatile collection of folded motifs. We argue that modified versions of the standard bases, some with features that resemble cofactors, could have facilitated that first moment in which early RNA molecules with catalytic capability began their evolutionary path toward self-replication.

Serum biomarkers in Duchenne muscular dystrophy (DMD) may provide deeper insights into disease pathogenesis, suggest new therapeutic approaches, serve as acute read-outs of drug effects, and be useful as surrogate outcome measures to predict later clinical benefit. In this study a large-scale biomarker discovery was performed on serum samples from patients with DMD and age-matched healthy volunteers using a modified aptamer-based proteomics technology. Levels of 1,125 proteins were quantified in serum samples from two independent DMD cohorts: cohort 1 (The Parent Project Muscular Dystrophy-Cincinnati Children's Hospital Medical Center), 42 patients with DMD and 28 age-matched normal volunteers; and cohort 2 (The Cooperative International Neuromuscular Research Group, Duchenne Natural History Study), 51 patients with DMD and 17 age-matched normal volunteers. Forty-four proteins showed significant differences that were consistent in both cohorts when comparing DMD patients and healthy volunteers at a 1% false-discovery rate, a large number of significant protein changes for such a small study. These biomarkers can be classified by known cellular processes and by age-dependent changes in protein concentration. Our findings demonstrate both the utility of this unbiased biomarker discovery approach and suggest potential new diagnostic and therapeutic avenues for ameliorating the burden of DMD and, we hope, other rare and devastating diseases.

Using modified nucleotides and selecting for slow off-rates in the SELEX procedure, we have evolved a special class of aptamers, called SOMAmers (slow off-rate modified aptamers), which bind tightly and specifically to proteins in body fluids. We use these in a novel assay that yields 1:1 complexes of the SOMAmers with their cognate proteins in body fluids. Measuring the SOMAmer concentrations of the resultant complexes reflects the concentration of the proteins in the fluids. This is simply done by hybridization to complementary sequences on solid supports, but it can also be done by any other DNA quantification technology (including NexGen sequencing). We use measurements of over 1000 proteins in under 100 μL of serum or plasma to answer important medical questions, two of which are reviewed here. A number of bioinformatics methods have guided our discoveries, including principal component analysis. We use various methods to evaluate sample handling procedures in our clinical samples and can identify many parameters that corrupt proteomics analysis.

Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of Escherichia coli , σ 70 . AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a novel helical fold for the protomer. Furthermore, the AsiA protomer, surprisingly, contains a helix–turn–helix DNA binding motif, predicting a potential new role for AsiA. The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen/deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer. In addition, the residues that form the dimer interface are those that are involved in binding to σ 70 . The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to σ 70 , where an AsiA protomer is displaced from the dimer via the interaction of σ 70 with the same residues in AsiA that constitute the dimer interface.

Background: CT screening for lung cancer is effective in reducing mortality, but there are areas of concern, including a positive predictive value of 4% and development of interval cancers. A blood test that could manage these limitations would be useful, but development of such tests has been impaired by variations in blood collection that may lead to poor reproducibility across populations. Results: Blood-based proteomic profiles were generated with SOMAscan technology, which measured 1033 proteins. First, preanalytic variability was evaluated with Sample Mapping Vectors (SMV), which are panels of proteins that detect confounders in protein levels related to sample collection. A subset of well collected serum samples not influenced by preanalytic variability was selected for discovery of lung cancer biomarkers. The impact of sample collection variation on these candidate markers was tested in the subset of samples with higher SMV scores so that the most robust markers could be used to create disease classifiers. The discovery sample set (n = 363) was from a multi-center study of 94 non-small cell lung cancer (NSCLC) cases and 269 long-term smokers and benign pulmonary nodule controls. The analysis resulted in a 7-marker panel with an AUC of 0.85 for all cases (68% adenocarcinoma, 32% squamous) and an AUC of 0.93 for squamous cell carcinoma in particular. This panel was validated by making blinded predictions in two independent cohorts (n = 138 in the first validation and n = 135 in the second). The model was recalibrated for a panel format prior to unblinding the second cohort. The AUCs overall were 0.81 and 0.77, and for squamous cell tumors alone were 0.89 and 0.87. The estimated negative predictive value for a 15% disease prevalence was 93% overall and 99% for squamous lung tumors. The proteins in the classifier function in destruction of the extracellular matrix, metabolic homeostasis and inflammation. Conclusions: Selecting biomarkers resistant to sample processing variation led to robust lung cancer biomarkers that performed consistently in independent validations. They form a sensitive signature for detection of lung cancer, especially squamous cell histology. This non-invasive test could be used to improve the positive predictive value of CT screening, with the potential to avoid invasive evaluation of nonmalignant pulmonary nodules.

Backgound : Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with K d S (equilibrium dissociation constant) in the range of 1 pM to 1 nM. Methods and Results : The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers. Conclusions : Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.

The field of diagnostics is undergoing revolutionary changes. Historically, searching for new diagnostic analytes – biomarkers – has targeted single molecules with sufficient information content to...

We used limited trypsin digestion to determine the domain organization of theEscherichia coliRNA polymerase σ70subunit. Trypsin-resistant fragments containing σ70conserved region 2 (σ702), and carboxy-terminal fragments containing conserved regions 3 and 4 (σ703 – 4) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of σ70·σ702bound core RNA polymerase competitively with intact σ70. In contrast to σ702alone, the RNA polymerase holoenzyme formed with σ702specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the −10 promoter element (the Pribnow box). σ702also forms crystals that are suitable for X-ray analysis. σ703 – 4bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on σ70was further localized to within σ70[551 – 608], comprising σ conserved region 4.2.

DNA microarrays are currently playing a central role in biomarker discovery and in the development of diagnostics for personalized medicine. Our vision is a technology that enables proteomics in the same revolutionary way that DNA microarrays enabled nucleic acid-based omics, allowing simple, reliable, sensitive, accurate, quantitative, and highly multiplexed measurements for the discovery of protein biomarkers and the development of new diagnostics to transform personalized medicine. We recently reached an important milestone, making unbiased protein biomarker discovery routine and fast. Microarrays played a prominent role in our experiments and will be central to the ongoing evolution of our platform. Our technology is powered by a new class of single-stranded DNA-based protein affinity binding reagents we call SOMAmers—slow off-rate modified aptamers. SOMAmers have a dual nature that is essential in our work: under normal conditions (e.g., physiologic in serum), SOMAmers fold into specific shapes that bind target proteins with high affinity (sub-nM K d), but when SOMAmers are denatured, they can be detected and quantified by hybridizing to a standard DNA microarray.

Background: Malignant pleural mesothelioma (MM) is an aggressive, asbestos-related pulmonary cancer that is increasing in incidence. Because diagnosis is difficult and the disease is relatively rare, most patients present at a clinically advanced stage where possibility of cure is minimal. To improve surveillance and detection of MM in the high-risk population, we completed a series of clinical studies to develop a noninvasive test for early detection. Methodology/Principal Findings: We conducted multi-center case-control studies in serum from 117 MM cases and 142 asbestos-exposed control individuals. Biomarker discovery, verification, and validation were performed using SOMAmer proteomic technology, which simultaneously measures over 1000 proteins in unfractionated biologic samples. Using univariate and multivariate approaches we discovered 64 candidate protein biomarkers and derived a 13-marker random forest classifier with an AUC of 0.9960.01 in training, 0.9860.04 in independent blinded verification and 0.9560.04 in blinded validation studies. Sensitivity and specificity at our pre-specified decision threshold were 97%/92% in training and 90%/95% in blinded verification. This classifier accuracy was maintained in a second blinded validation set with a sensitivity/ specificity of 90%/89% and combined accuracy of 92%. Sensitivity correlated with pathologic stage; 77% of Stage I, 93% of Stage II, 96% of Stage III and 96% of Stage IV cases were detected. An alternative decision threshold in the validation study yielding 98% specificity would still detect 60% of MM cases. In a paired sample set the classifier AUC of 0.99 and 91%/94% sensitivity/specificity was superior to that of mesothelin with an AUC of 0.82 and 66%/88% sensitivity/specificity. The candidate biomarker panel consists of both inflammatory and proliferative proteins, processes strongly associated with asbestos-induced malignancy. Significance: The SOMAmer biomarker panel discovered and validated in these studies provides a solid foundation for surveillance and diagnosis of MM in those at highest risk for this disease.

Infection of Escherichia coli by bacteriophage T4 induces a mRNA ribonuclease activity that shows specificity for cleavage within the sequence GGAG. Substrates of the activity in vivo include a number of phage mRNAs that are cleaved at GGAGs within the Shine/Dalgarno domains of their translation initiation regions. Induction of the ribonuclease depends on the product of the T4 gene regB. We describe here the overproduction and extensive purification of the RegB protein. RegB precisely co-purifies with an activity that cleaves within the sequence GGAG in oligonucleotide and polynucleotide RNAs and is therefore likely to constitute the sequence-specific catalytic component of the observed activity. We further report that the low cleavage rate observed with our preparations of purified RegB is substantially increased (1-2 orders of magnitude) by the addition of E. coli ribosomal protein S1. We discuss the implications of this observation for the mechanism of action of the RegB ribonuclease in vitro and in vivo.

The asiA gene of bacteriophage T4 encodes a 10-kDa peptide which binds strongly in vitro to the sigma 70 subunit of Escherichia coli RNA polymerase, thereby weakening sigma 70-core interactions and inhibiting sigma 70-dependent transcription. To assess the physiological role of this protein, we have introduced an amber mutation into the proximal portion of the asiA gene. On suppressor-deficient hosts, this mutant phage (amS22) produces minute plaques and exhibits a pronounced delay in phage production. During these mutant infections, T4 DNA synthesis is strongly delayed, suggesting that the AsiA protein plays an important role during the prereplicative period of phage T4 development. The kinetics of protein synthesis show clearly that while T4 early proteins are synthesized normally, those expressed primarily via the middle mode exhibit a marked inhibition. In fact, the pattern of protein synthesis after amS22 infection resembles greatly that seen after infection by amG1, an amber mutant in motA, a T4 gene whose product is known to control middle mode RNA synthesis. The amber mutations in the motA and asiA genes complement, both for phage growth and for normal kinetics of middle mode protein synthesis. Furthermore, primer extension analyses show that three different MotA-dependent T4 middle promoters are not recognized after infection by the asiA mutant phage. Thus, in conjunction with the MotA protein, the AsiA protein is required for transcription activation at T4 middle mode promoters.

Single protein biomarkers measured with antibody-based affinity assays are the basis of molecular diagnostics in clinical practice today. There is great hope in discovering new protein biomarkers and combinations of protein biomarkers for advancing medicine through monitoring health, diagnosing disease, guiding treatment, and developing new therapeutics. The goal of high-content proteomics is to unlock protein biomarker discovery by measuring many (thousands) or all (∼23,000) proteins in the human proteome in an unbiased, data-driven approach. High-content proteomics has proven technically difficult due to the diversity of proteins, the complexity of relevant biological samples, such as blood and tissue, and large concentration ranges (in the order of 10(12) in blood). Mass spectrometry and affinity methods based on antibodies have dominated approaches to high-content proteomics. For technical reasons, neither has achieved adequate simultaneous performance and high-content. Here we review antibody-based protein measurement, multiplexed antibody-based protein measurement, and limitations of antibodies for high-content proteomics due to their inherent cross-reactivity. Finally, we review a new affinity-based proteomic technology developed from the ground up to solve the problem of high content with high sensitivity and specificity. Based on a new generation of slow off-rate modified aptamers (SOMAmers), this technology is unlocking biomarker discovery.

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.

The anti-sigma factor AsiA effects a change in promoter specificity of the Escherichia coli RNA polymerase via interactions with two conserved regions of the sigma(70) subunit, denoted 4.1 and 4.2. Free AsiA is a symmetrical homodimer. Here, we show that AsiA is monomeric when bound to sigma(70) and that a subset of the residues that contribute to the homodimer interface also contributes to the interface with sigma(70). AsiA interacts primarily with C-terminal sections of regions 4.1 and 4.2, which show remarkable sequence similarity. An AsiA monomer can simultaneously, and apparently cooperatively, bind both isolated regions 4.1 and 4.2 at preferred, distinct subsites, whereas region 4.1 alone or region 4.2 alone can interact with either subsite. These results suggest structural and functional plasticity in the interaction of AsiA with sigma(70) and support the notion of discrete roles for regions 4.1 and 4.2 in transcription regulation by AsiA. Furthermore, we show that AsiA inhibits recognition of the -35 consensus promoter element by region 4 of sigma(70) indirectly, as the residues on region 4 responsible for AsiA binding are distinct from those involved in DNA binding. Finally, we show that AsiA must directly disrupt the interaction of region 4 with the RNA polymerase beta subunit flap domain, resulting in a distance change between region 2 and region 4 of sigma(70). Thus, a new paradigm for transcription regulation by AsiA is emerging, whereby the distance between the DNA binding domains in sigma(70) is regulated, and promoter recognition specificity is modulated, by mediating the interactions of the sigma region 4 with the beta subunit flap domain.

The association of the bacteriophage T4-encoded AsiA protein with the final sigma(70) subunit of the Escherichia coli RNA polymerase is one of the principal events governing transcription of the T4 genome. Analytical ultracentrifugation and NMR studies indicate that free AsiA is a symmetric dimer and the dimers can exchange subunits. Using NMR, the mutual recognition sites on AsiA and final sigma(70) have been elucidated. Residues throughout the N-terminal half of AsiA are involved either directly or indirectly in binding to final sigma(70) whereas the two highly conserved C-terminal regions of final sigma(70), denoted 4.1 and 4.2, constitute the entire AsiA binding domain. Peptides corresponding to these regions bind tightly to AsiA individually and simultaneously. Simultaneous binding promotes structural changes in AsiA that mimic interaction with the complete AsiA binding determinant of final sigma(70). Moreover, the results suggest that a significant rearrangement of the dimer accompanies peptide binding. Thus, both conserved regions 4.1 and 4.2 are intimately involved in recognition of AsiA by final sigma(70). The interaction of AsiA with 4.1 provides a potential explanation of the differential abilities of DNA and AsiA to bind to free final sigma(70) and a mechanistic alternative to models of AsiA function that rely on binding to a single site on final sigma(70).

In bacterial transcription, diverse sigma-family promoter recognition proteins compete for a common RNA polymerase core. Bacteriophage T4 infection ultimately reduces this competition to a duel between activated viral middle and enhanced late transcription, involving two sigma proteins, two phage-encoded activator proteins and two phage-specific co-activators. This competition has been analyzed in vitro, and the relative abundances in T4-infected Escherichia coli of the participating proteins have been measured. Activated late transcription holds an advantage over activated middle transcription, especially at higher ionic strength. This advantage is further compounded by ADP-ribosylation of the RNA polymerase alpha subunits, and by the phage-specific, RNA polymerase core-bound RpbA subunit. The largest contribution to the middle-late competition is made by gp55, the late sigma factor, but not enough of gp55 is produced during T4 infection to shut off middle transcription by direct competition with sigma(70). AsiA, the originally identified anti-sigma protein is not a major determinant of middle-late competition.

The bacteriophage T4 encodes proteins that are responsible for tightly regulating mRNA synthesis throughout phage development in Escherichia coli . The three classes of T4 promoters (early, middle, and late) are utilized sequentially by the host RNA polymerase as a result of phage-induced modifications. One such modification is the tight binding of the T4 AsiA protein to the σ 70 subunit of the RNA polymerase. This interaction is pivotal for the transition between T4 early and middle transcription, since it both inhibits recognition of host and T4 early promoters and stimulates T4 middle mode synthesis. The activation of T4 middle transcription also requires the T4 MotA protein, bound specifically to its recognition sequence, the “Mot box,” which is centered at position −30 of these promoters. Accordingly, the two T4 proteins working in concert are sufficient to effectively switch the transcription specificity of the RNA polymerase holoenzyme. Herein, we investigate the mechanism of transcription activation and report that, while the presence of MotA and AsiA increases the initial recruitment of RNA polymerase to a T4 middle promoter, it does not alter the intrinsic stability of the discrete complexes formed. In addition, we have characterized the RNA polymerase-promoter species by UV laser footprinting and followed their evolution from open into initiating complexes. These data, combined with in vitro transcription assays, indicate that AsiA and MotA facilitate promoter escape, thereby stimulating the production of full-length transcripts.

The AsiA protein of bacteriophage T4 binds to the sigma70 subunit of Escherichia coli RNA polymerase and plays a dual regulatory role during T4 development: (i) inhibition of host and phage early transcription, and (ii) coactivation of phage middle-mode transcription, which also requires the T4 DNA binding transcriptional activator, MotA. We report that the interaction between AsiA and sigma70 occurs with a 1:1 stoichiometry. When preincubated with RNA polymerase, AsiA is a potent inhibitor of open complex formation at the lac UV5 promoter, whereas it does not perturb preformed open or intermediate promoter complexes. DNase I footprinting and electrophoretic mobility shift analyses of RNA polymerase-DNA complexes formed at the T4 early promoter P15.0 show that AsiA blocks the initial RNA polymerase binding step that leads to the formation of specific closed promoter complexes. A contrasting result is obtained on the T4 middle promoter PrIIB2, where AsiA stimulates the formation of both closed complexes and open complexes. Therefore, we propose that AsiA modulates initial DNA binding by the RNA polymerase, switching promoter usage at the level of closed complex formation.

The chicken β-tropomyosin pre-mRNA is spliced in a tissue-specific manner. Internal exons 6B and 6A are specifically used in skeletal muscle and non-skeletal muscle cells, respectively. Pre-mRNA secondary structure around exon 6B has been shown to be part of the mechanism that inhibits exon 6B to 7 splicing in HeLa nuclear extract. We analyse the influence of pre-mRNA folding on the different steps of spliceosome assembly under different conditions. At 3 mM MgCl 2 , conditions that favour RNA structure formation, the interactions of U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) with the pre-mRNA are all affected. The study of several mutants destabilising some proposed stem-loop structures shows that the in vitro splicing activation is correlated with an increased binding of snRNPs on pre-mRNA molecules. At 1 mM MgCl 2 , conditions that allow a partial relaxation of the inhibitory structure, U1 snRNP binding on exon 6B 5′ splice site occurs very efficiently. Nonetheless,if this first step of spliceosome assembly is derepressed, U2, U4, U5 and U6 snRNP interaction processes remain inhibited. Altogether, these results suggest that the choice between exon 6A and 6B donor sites is a complex process not simply directed by a difference in the efficiency of interaction between U1 snRNP and alternative 5′ splice sites.

A previous study has identified a C-->U mutation at position -3 in the 3' splice site of intron 10 of the phenylalanine hydroxylase pre-mRNA in a patient with phenylketonuria. In vivo, this mutation induces the skipping of the downstream exon. This result is puzzling because both CAG and UAG have been reported to function equally as 3' splice sites. In this report, we show that the C-->U mutation affects predominantly the first step of the splicing reaction and that it blocks spliceosome assembly at an early stage. The 3' region of the phenylalanine hydroxylase intron 10 has two unusual characteristic features: multiple potential branch sites and a series of four guanosine residues, which interrupt the polypyrimidine tract at positions -8 to -11 from the 3' splice site. We show that the mutation precludes the use of the proximal branch site, while having no effect on the remote one. We also show that in the UAG transcript, the four guanosine residues inhibit the splicing of intron 10. The substitution of these purine residues by one cytosine residue, regardless of the position, increases the splicing efficiency of the mutant UAG precursor while having no effect on the wild-type CAG precursor. Substituting the four purine residues by four pyrimidines relieves the inhibition and rescues the use of the proximal branch site. These results demonstrate that according to the context, the C and U nucleotides preceding the AG are not equivalent for the splicing reaction.

We have identified a nucleolytic activity in Escherichia coli infected by bacteriophage T4 that introduces cuts in the ribosome binding site of at least two T4 mRNAs. Cutting takes place specifically in the GGAG sequences that are complementary to the 3' end of 16S rRNA (Shine-Dalgarno sequence). The nature of this nucleolytic cut has been investigated by reverse transcriptase mapping, anti-mRNA mapping, utilization of the vaccinia virus guanylyltransferase, and labeling by polynucleotide kinase. We have compared the sequences of target mRNAs with an mRNA of similar sequence but that is not a substrate for the nuclease. This allowed us to narrow down the possibilities for sequence elements that determine nuclease recognition. We hypothesize that this nuclease plays a physiological role in the inhibition of expression of a class of phage proteins.

The effect of the E. coli termination factor rho on middle mode and anti-late T4 transcription was examined. RNA from CAM-treated rho ts-infected cells contains only about 25% of the normal T4 gene 43 RNA levels, despite the fact that the DE content of this RNA is qualitatively normal. Gene 43 RNA is not found during the early period of infection of rho+ bacteria by mot− bacteriophage, but about 20–25% normal levels are found when mot− phage infect rho ts hosts. Thus gene 43 is transcribed in two modes: a major middle mode which uses mot as an initiation cofactor and a minor early mode which seems to need mot expression to help overcome rho action. Apparently, the middle mode RNA from gene 43 is translated much more efficiently than is the early RNA transcribed from this gene. CAM RNA from rho ts infections contains nearly as much anti-late RNA as does normal RNA (without antibiotic) from these cells. Thus anti-late synthesis, which is ordinarily mot dependent, becomes mot independent if there is no rho activity in the hosts cells. These data along with experiments on an in vitro system for middle mode expression ( C. Thermes, P. Daegelen, V. de Franciscis, and E. Brody (1976) Proc. Nat. Acad. Sci. USA 73, 2569–2573) are consistent with a model in which the mot product binds to DNA where it acts either as a melting protein or as a gyrase. This DNA change leads to changed interactions both with rho and RNA polymerase.

The effect of T4 ghosts on T4 development has been examined by analysis of early RNA species and polyacrylamide gel electrophoresis of proteins. Ghosts interfere with early development from the moment of infection. The transition from immediate to delayed early RNA is slowed down by ghosts as is the appearance and disappearance of early proteins. We have concluded that the delay seen in the immediate to delayed early transition is not a direct effect of ghost alteration of a membrane site for this transition. Ghosts also have an effect on the ability of T4 bacteriophage to inhibit host protein and RNA synthesis. Host protein bands are seen as late as 10 min after simultaneous ghost-phage infection, whereas such bands are no longer visible 3 min after normal phage infection.

The synthesis of five specific bacteriophage T4 proteins was measured following transcription and translation in vitro of native and sheared bacteriophage T4 DNA. T4 DNA of decreased molecular weight was found to direct preferentially the synthesis of those proteins whose genes are transcribed earliest by RNA polymerase in vitro . The results support a model for T4 DNA transcription in which the time interval between initiation of RNA synthesis and elongation of a specific gene product reflects the physical distance between a promoter and that gene.

The synthesis of different early T4 transcripts by E. coli RNA polymerase in vitro can be dissociated in two ways: (1) when T4 DNA is mechanically degraded, it selectively loses the ability to serve as a template for the transcription of delayed early T4 RNA; (2) when initiation of RNA synthesis on intact T4 DNA is limited to a very short time interval immediate early T4 RNA is synthesized at once but delayed early polynucleotide sequences appear only after a time lag. We conclude that initiation sites for T4 early RNA synthesis are closer to immediate early than to delayed early segments of the T4 template and that immediate early and delayed early segments of the T4 chromosome are interspersed. We propose that this interspersing is significant for the programming of transcription during T4 development.

T1 deoxyribonucleic acid (DNA) infection of spheroplasts was characterized by the following. A small number of the DNA molecules initiated infectious centers, and a small number of the spheroplasts were infected by T1 DNA. Once a favorable encounter of T1 DNA with spheroplast occurred, a minimum of 20 to 30 min was required for T1 DNA to enter the spheroplast. The mature T1 particles produced in the infection of spheroplasts by T1 DNA were released in a burst, but the average burst size was quite small compared with a normal burst of the phage-infected bacteria. T1 DNA preparations, capable of causing viral growth in spheroplasts, did not require detectable amounts of protein for infectivity, were homogeneous in band and boundary sedimentation, and had a guanine plus cytosine content of 48% and a minimal molecular weight of 35 × 10 6 . Denatured T1 DNA, like denatured λDNA, did not infect spheroplasts. Renatured T1 DNA was not infectious; this was in marked contrast to renatured λDNA.

We have analyzed transcription in vitro in mixtures of ethylene glycol and water. We find two major effects of this solvent on T4 transcription, both of which are reversible on dilution out of the solvent: (a) only very short (10% normal length) T4 chains are formed; (b) a small percentage of initiation errors are made; these include initiation on the “wrong” strand and initiation on the “wrong” site of the correct strand. The ratio of GTP to ATP initiation is also increased in this solvent and it may be that all faulty initiation is done with GTP. This faulty initiation seems to derive, Like normal initiation, from a σ-dependent reaction on native T4 DNA templates, and is thus distinguished from that faulty initiation which derives from core synthesis on damaged or denatured templates. Analysis of the products of T4 transcription in vitro in ethylene glycol suggests that this reagent has a different effect on RNA chains synthesized from promoter sequences than it does on chains initiated from non-promoter sequences. Those chains which start at promoters show a very strong asymmetry, while those chains which start from non-promoter sequences are completely symmetrical despite their σ-dependence. Transcription of poly(dG) · poly(dC) and poly(dA) · poly(dT) is relatively little changed in 30% ethylene glycol. The synthesis of short poly(G) chains with frequent terminations and re-initiations takes place in this solvent just as it does in aqueous buffers. The selective synthesis of polypurines from these templates is not perturbed, even in 40% ethylene glycol. The inherent asymmetry of promoters in ethylene glycol is considered from the point of view of conformational changes undergone by DNA and homopolymers in ethylene glycol solutions.

Radioactive proteins have been synthesized in vitro from a coupled transcription-translation system primed with bacteriophage T4 DNA. The labeled proteins were chromatographed on RNA polymerase-Sepharose affinity columns in order to identify a protein which comigrates with the 10-kDa anti-σ subunit of T4-modified RNA polymerase. When we primed the in vitro system with specific restriction fragments of T4 DNA, we found that there seemed to be two widely separated genes which code for this protein. The products of these two genes were compared by two-dimensional electrophoresis; they were found to have different charges, even though they had the same molecular weight and strong affinity for RNA polymerase. One of these proteins exists in a form which has the same charge as the 10-kDa subunit isolated from purified T4-modified RNA polymerase. We report a preliminary mapping experiment within the 22.5-kb fragment harboring this gene.

We have constructed a bacteriophage T4 isar which is deleted in the rII and D2 regions and simultaneously allows cells to be superinfected after a primary infection. After infecting with this phage, we add chloramphenicol to the cells, and after a short period we introduce the rII and D2 regions into the cell by superinfection with a phage carrying these regions. Transcription of the rII and D2 regions takes place very rapidly after superinfection. We conclude that there is a stable, diffusible T4 effect which overcomes the restriction of T4 transcription seen when infection is done in the presence of chloramphenicol. The kinetics of rIIA and rIIB synthesis are much more rapid when these genes are first introduced into a T4 isar infected cell than in a normal infection. We think that a promoter close to rIIA is used in superinfection of T4isar infected cells; this promoter is different from the promoter usually used for the synthesis of rIIA RNA.

We have used ts lethal mutants in Escherichia coli termination factor rho to examine its role in T4 development. The rho ts mutation has been transduced into E. coli B from E. coli K12. This mutation has a number of different properties in the E. coli B genetic background, but the overall effect on T4 regulation seems to be the same. Rho-defective bacteria produce T4 after infection but the burst size is lower than in rho + host cells. We have found that two protein bands appear after T4 + infections of rho ts , but not rho + , bacteria. One of these is gp alt and the other is gp 7. These proteins seem to be expressed in a different mode than the late mode in rho + cells. Their appearance is sensitive to a mutation in the T4 mot gene. When T4 + infects E. coli rho + bacteria in the presence of chloramphenicol (CAM), only a subfraction of early RNA is found. This RNA comes from promoter proximal portions of early polycistronic transcription units and is called immediate early RNA (IE). CAM-sensitive RNA is defined as delayed early (DE) RNA. CAM RNA from rho ts bacteria has almost normal amounts of DE RNA. Thus, CAM-induced polarity, which limits T4 transcription to IE RNA, is mediated by rho factor.

Tissue specific splicing of pre-messenger RNA is an important mechanism of regulating metazoan gene expression. There are scores of examples of single species of pre-messenger RNA which are synthesized in various tissues or in one tissue at various stages of development, but which are spliced to give slightly different collections of exons in each tissue or at each stage of development. We wish to contrast such tissue specific splicing to alternative splicing: in the latter, various collections of exons are spliced from the same pre-messenger RNA, but these different splicing events take place in the same cell at the same time. Alternative splicing, then, generates diversity, but not necessarily regulation. In this article, we shall argue that alternative splicing was a characteristic of all primitive splicing systems. We shall further argue that during evolution, the precision of splicing was increased and that this led to two different results. One was constitutive splicing, in which one and only one 5′ end of an intron is spliced to a unique 3′ end of this intron. The other was tissue specific splicing, in which the original ambiguity of primitive splicing was harnessed to the demands of differentiating tissues in metazoans.