Creation of libraries with long ORFs by polymerization of a microgene

We describe a novel method for constructing pools of DNA sequences that encode large proteins with molecular diversity. Sets of primer pairs that form 8 to 10 complementary base pairs in the 3' region and have double mismatch pairs at their 3'-OH ends were designed so that primer dimers recreated short stretches of DNA (microgenes) devoid of termination codons. Cycles of denaturation and elongation reactions with a pair of primers, four dNTPs, and 3'-5' exo+ thermostable DNA polymerase gave head-to-tail polymers of the primer dimer unit (microgene) whose sizes exceeded 12 kb. No template was required in this reaction, but mismatched nucleotides at 3'-OH ends of the primers were critical for efficient polymerization. At end-joining junctions of a microgene, nucleotide insertions and deletions randomly occurred, resulting in combinatorial libraries of three reading frames from a single microgene. Further molecular diversity could be incorporated by using a mixture of primers. The resultant polymers have long ORFs whose products have a repetitious nature that could facilitate the formation of higher structures of translated products. Thus, microgene polymers may be used as a source of libraries for in vitro protein evolution experiments. Ligation of a microgene is apparently related to the nonhomologous recombination of double-strand breaks in DNA that has been shown to be catalyzed by DNA polymerases. We named this polymerization reaction the "microgene polymerization reaction."     

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.     

Murine NIMA-related kinases are expressed in patterns suggesting distinct functions in gametogenesis and a role in the nervous system

NIMA protein kinase is a major regulator of progression into mitosis in Aspergillus nidulans. Dominant negative forms of NIMA protein prevent entrance into mitosis in HeLa cells, suggesting that mammals have a similar pathway. We have reported previously the isolation of a murine NIMA-related kinase, designated Nek1, and more recently several additional NIMA-related human kinases have been cloned. The existence of several mammalian NIMA-related genes raises the questions of whether the different mammalian members have redundant, overlapping or distinct functions, and whether these functions are related to the role of NIMA in controlling mitosis. To address these questions we have studied the expression patterns of the different murine nek genes. To this end, we isolated a murine nek2 cDNA and compared its patterns of expression, during both gametogenesis and embryogenesis, to those of nek1. Both genes were highly expressed in developing germ cells, albeit in distinct patterns. In both females and males, nek1 is expressed much earlier than nek2, suggesting only limited ability for functional redundancy. Surprisingly, a striking specificity of nek1 expression was found: high levels of nek1 RNA were observed in distinct regions of the nervous system, most notably in neurons of the peripheral ganglia. These patterns suggest that the different mammalian NIMA-related kinases participate in different phases of the meiotic process and may also have functions other than cell cycle control.     

The conclusions from an analysis of 9 approaches to determining the value of KT/Vur

OBJECTIVES: The polymerase chain reaction (PCR)-based method was used to obtain and sequence three H-2K and three H-2D mouse complementary DNAs (cDNA) of class I major histocompatibility complex (MHC) molecules. METHODS: Messenger RNA was isolated from Conconavalin A-activated splenocytes of C57BL/10 (H-2b), C3H (H-2k), and Balb/c (H-2d) mice. We designed H-2K- and H-2D-specific primers as well as a common downstream primer based on previously published mouse class I MHC sequences. Using the PCR method and selective primers we isolated and sequenced H-2Kb and H-2Db cDNAs of C57BL/10, H-2Kk and H-2k cDNAs of C3H, as well as H-2Kd and H-2Dd cDNAs of Balb/c strains. RESULTS: Analysis of the nucleotide sequences documented similarity between our three H-2K cDNA sequences and all mouse MHC class I sequences available in the GenBank. Similarly, our three H-2D sequences were homologous with all mouse class I MHC sequences deposited in the GenBank. Our H-2K and H-2D sequences were also identical to numerous published sequences. CONCLUSIONS: Using these mouse cDNAs, we plan to determine the localization of polymorphic in vivo immunogenic amino acids in class I MHC H-2K and H-2D alloantigens.     

Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction

We report a method for optimizing the specificity of product formation in the polymerase chain reaction (PCR). This technique is based on the use of dimethyl sulfoxide (DMSO) and takes into account primer Tm. The reduction in Tm by DMSO is directly correlated with renaturation temperature such that a DMSO gradient reflects a temperature gradient. We use this relationship to show that optimum product formation usually occurs at or within several degrees of the midpoint Tm of a given primer pair. We illustrate these correlations using three examples deriving PCR products from a human cDNA library, representing the casein kinase II alpha and beta subunits as well as the 5' untranslated region for the beta subunit. By following product formation as a function of renaturation temperature, we postulate rules for cycle design based on primer Tm. Implications for the use of degenerate primers are discussed.     

Partial inhibition of amplifications by primers of EHEC genes

Diagnostic value of multiplex polymerase chain reaction (PCR) was examined by using three primer pairs, specific for the common conserved region of stx1 and stx2, eae and an enterohaemolysin A gene (ehxA). The sensitivity in respect of each amplicon decreased with three exponents comparing to the individual PCR reactions. These PCR reactions were partially inhibited by the presence of certain additional primers. This inhibitory effect was template-concentration dependent, and was partially balanced by usage of increased amount of dNTP. Taq DNA polymerase in a range of 0.3-1.25 U/reaction did not influence the inhibition. The same inhibition was detected if the annealing temperature was changed from 48 degrees C to 57 degrees C. Pairs of EHEC primers inhibited a Salmonella enteritidis virulence-plasmid specific gene amplification, as well. Theoretical inhibiting effects were predicted by Primer Premier software but our observations can be sufficiently explained neither by the competitions between the specific and aspecific amplifications nor by the inhibition caused by dimerization of primers.     

Absence of mutations in the analysis of coding sequences of the entire transforming growth factor-beta type II receptor gene in sporadic human breast cancers

The transforming growth factor-beta (TGFbeta) binds the type II TGFbeta growth factor receptor (TGFbetaRII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the TGFbetaRII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of TGFbetaRII gene mutations, in this study, we examined 36 sporadic breast cancers. We previously produced eight intron based primer pairs for mutational analysis of the entire coding region of the TGFbetaRII gene. Using these primers, we developed protocols for polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of PCR products from genomic DNA samples of 36 breast cancer patients and we tested them for microsatellite instability (MI) at eight microsatellite loci. One case demonstrated MI (2.8%) and we found no mutations. These and other recent data indicate that TGFbetaRII mutations are essentially confined to colon and gastric cancers with MI. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.     

Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus

It has been shown that vaccination with two low molecular mass excretory secretory (ES) antigens of 15 and 24 kDa, respectively, afforded a substantial degree of protection against Haemonchus contortus to sheep. In vitro cultivation of the parasite usually yields a limited amount of these proteins and therefore, recombinant DNA technology was employed to clone the cDNAs encoding the ES proteins of interest and to express them in a convenient vector system. The N-terminal amino acid sequences of the two ES products were determined. Specific 5' primers were used in combination with an oligo (dT) 3' primer to amplify the appropriate cDNAs by polymerase chain reaction (PCR). A lambda ZAPII cDNA library was constructed from mRNA of L5 larvae and subsequently screened with the PCR products. The full length clone of the 15 kDa ES protein coded for a 17.2 kDa precursor molecule of 148 amino acids with a signal peptide of 30 amino acids. The full length clone of the 24 kDa ES protein coded for a 24.6 kDa precursor protein of 222 amino acids with a leader sequence of 19 residues. The expression of both ES products appeared to be developmentally regulated; mRNA encoding occurs only in the parasitic life stages. A cDNA of each ES protein was sub-cloned, without the leader sequence, into a pQE9 expression vector. Both purified recombinant proteins were recognized by sera from H. contortus hyperimmunised sheep as judged by immunoblot analysis, suggesting that antigenic determinants were also present on the recombinant proteins.     

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum

The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo(R) v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo(R) 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.     

Regulation of the anaphase-promoting complex/cyclosome by bimAAPC3 and proteolysis of NIMA

Surprisingly, although highly temperature-sensitive, the bimA1(APC3) anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1(APC3) is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34(cdc2) kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1(APC3)-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1(APC3) double mutant arrests in a mitotic state with very high p34(cdc2) H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1(APC3) mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1(APC3). The bimA1(APC3) mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.     

Effect of extracapsular extension on cervical recurrence and the survival of patients with laryngeal tumors

Distinction between benign and malignant T-cell lymphoproliferative diseases can be difficult using morphological criteria. Using multiplex polymerase chain reaction system we have tested a series of patients with various lymphoproliferative disorders to detect clonal T-lymphocyte populations. Results show that clonal amplification products were obtained from all 10 patients with T-cell lymphoproliferative disorders while the amplification of DNA samples from B-cell neoplasms and normal individuals revealed polyclonal amplification products. By splitting the multiplex primer mix, the patient specific T-cell receptor gamma rearrangement was determined: five out of ten patients showed the exclusive presence of a single T-cell receptor gamma gene rearrangement. Three patients exhibited two rearranged T-cell receptor gamma genes, while in two patients positive reactions were obtained with three pairs of primers for variable and joining segments. Molecular analysis of rearranged T-cell receptor genes by multiplex polymerase chain reaction represents a useful and rapid tool for confirming diagnosis, to determine the extent of disease and to monitor the response to therapy.     

Differentiation of condylomata acuminata from pseudocondyloma by using multiple primer pairs polymerase chain reaction

Condylomata acuminata were differentiated from pseudocondyloma by using multiple primer pairs polymerase chain reaction (PCR) technique. The paraffin-embedded tissues of 95 cases of condylomata acuminata and 33 cases of pseudocondyloma were detected for HPV DNA. Results show that HPV 6/11 DNA are found in 86/95 (90.5%) cases of condylomata acuminata, and no HPV DNA is detected in 33 cases of pseudocondyloma. The results suggest that pseudocondyloma is not associated with HPV. Multiple primer pairs PCR technique is a preferable method for differentiating condylomata acuminata from pseudocondyloma.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Identification of a novel receptor tyrosine kinase expressed in acute myeloid leukemic blasts

Using the polymerase chain reaction with degenerate oligonucleotides derived from conserved motifs within the catalytic kinase domain of protein tyrosine kinases, and RNA extracted from embryonic stem cells, sequences that encode a segment of the kinase domain of several potentially novel receptor tyrosine kinases (RTKs) have been identified. One of these was selected for further study because in Northern analysis it hybridized to RNA from multipotential hematopoietic cell lines, but not from lines representative of lineage-committed cells. A cDNA for this receptor, designated developmental tyrosine kinase (DTK), was isolated and encodes a protein with structural similarities to AXL. Together these receptors form a new class of RTK. DTK is expressed in a number of human leukemic cell lines, and in the blasts of 6 of 11 patients with acute myeloid leukemia (AML) analyzed. The structure of DTK suggests that it may function as a cell adhesion molecule, and mediate cell-to-cell or cell-matrix interactions between hematopoietic cells and their respective microenvironments.     

Gene-specific universal mammalian sequence-tagged sites: application to the canine genome

We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail: CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB, and RB1. We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as "anchored reference loci" for the development of mammalian genetic maps [O'Brien, S. J., et al., Nat. Genet. 3:103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets "universal mammalian sequence-tagged sites" because they should be useful for many mammalian genome projects.     

GeneUp: a program to select short PCR primer pairs that occur in multiple members of sequence lists

A computer program is presented that selects a small set of short primer pairs for PCR to sample all the sequences in a user-specified list of mRNAs. Such primer pairs could be used to increase the probability of sampling mRNAs of particular interest in differential display and to generate simplified hybridization probes for DNA chips or arrays. The program uses simulated PCR to find pairs of primers that sample more than one sequence in the list. A small set of such primer pairs is selected that give maximal coverage of the sequences in the list. Primer pairs are excluded that: (i) generate simulated PCR products of the same size from a number of sequences in the list, (ii) can easily form primer dimers, (iii) are outside a specified range of G + C content or (iv) occur in another list of undesirable sequences, such as rRNAs and Alu repeats. Five lists consisting of from 48-285 cDNA sequences were used to test the program. A small number of pairs of primers, 8-10 bases in length, were selected that fit the above criteria and that generate one or more simulated PCR products in all or most of the cDNAs in each list.