Strand cDNA Synthesis Research Articles

ウシ初期はい内における複数の遺伝子発現に関する検討（アンチセンスＲＮＡ‐ＲＴ‐ＰＣＲ法）

We examined whether we can detect multiple gene expressiom in single early bovine embryos by using antisense RNA (aRNA) and RT-PCR (aRNA-RT-PCR). Embryos, from the 2-cell stage to the blastocyst stage, were produced by the IVM-IVF method, cryopreserved and used as material for aRNA synthesis. Antisense RNA solution was yielded after reverse-transcription (RT) with a synthetic primer containing the T7 RNA polymerase binding site, double strand cDNA synthesis and transcription by the T7 RNA polymerase. For the reverse transcription of aRNA (aRNA-RT), we used a 5 μl of total 100 μl aRNA solution (1/20) and either sense primer or an antisense one encoding bovine beta-actin (bActin). After the aRNA-RT and PCR, the presence of the expected products was confirmed by electrophoresis. We confirmed the bActin products when the sense primer was used in aRNA-RT, but we did not confirm the products with the antisense one. These results indicate that we could detect multiple gene expression in a single early bovine embryo by means of aRNA-RT-PCR.

The Mauriceville and Varkud plasmids: primitive retroelements found inNeurosporamitochondria

The Mauriceville and closely related Varkud plasmids are small circular DNAs (3.6 and 3.7 kb, respectively) found in the mitochondria of certain Neurospora spp. strains isolated from nature. The plasmids replicate via reverse transcription and appear to be primitive retroelements that may be related to the early ancestors of retroviruses. Recent studies have shown that the plasmid reverse transcriptase closely resembles certain viral RNA-dependent RNA polymerases in initiating (−) strand cDNA synthesis de novo (i.e., without a primer) at a tRNA-like structure at the 3′ end of the plasmid transcript. The plasmid reverse transcriptase can also use DNA or RNA primers and can carry out template-switching reactions that lead to the generation of suppressive mutant plasmids or the integration of the plasmids into mitochondrial DNA. The characteristics of the plasmids and their reverse transcription mechanism suggest an evolutionary connection between RNA and DNA replication and raise the possibility that the plasmids are related to the earliest DNA-based life forms that emerged at the time of transition from an RNA to a DNA world. Key words: DNA synthesis, evolution, retrovirus, reverse transcriptase, RNA virus.

Cellular deformation reversibly depresses RT-PCR detectable levels of bone-related mRNA

Osteoblastic cells respond to mechanical stimuli with alterations in proliferation and/or phenotypic expression. In some cases, these responses occur within only a few applications of stimuli (i.e. ‘cycle-dependent trigger response’) rather than in a dose-dependent manner. To explore potential mechanisms of the cycle dependent trigger response, we raised the following questions: (1) Does strain of bone cells alter gene expression; if so, how quickly does it occur and how long does it last? (2) Are alterations in message level strain magnitude dependent? (3) Are alterations in steady-state message levels cycle dependent? Cultures were evaluated for osteocalcin mRNA one week following a daily stretch application at four stretch magnitudes and four cycle numbers and compared to nonstretched controls. Steady state mRNA message was ascertained prior to and at 10, 20, 30, 60, 120, 180, and 240 min following initiation of stretch. Following mRNA isolation, first strand cDNA synthesis was performed and fluorometrically quantitated. A reverse transcriptase based PCR (RT-PCR) approach allowed assessment of osteocalcin mRNA levels from microcultures (50,000 cells per 10 μl culture or 5000 cells mm 2) of rat calvarial osteoblasts. Optimized PCR was performed using primers to the bone specific protein, osteocalcin (OC) and two ‘housekeeping’ genes, β-actin and GAP-DH. PCR products were separated on 4% agarose gels and band intensities digitized with relative quantitation based on internal standards in each gel. The lowest magnitude of stretch (−1 KPa) at 1800 cycles per day reproducibly depressed message for osteocalcin, but not β-actin when assayed immediately following the cessation of strain application. By three hours following the initiation of stretch, message levels returned to control values. At the time of stretch cessation, the 1800 cycle stretch regimen diminished ( p < 0.0001) steady-state osteocalcin message independently of the four stretch magnitudes. Stretch for 300 cycles failed to depress ( p = 0.05) osteocalcin message cultures at any time, but 600 cycles depressed message by 30 min. By one and two hours, cultures stretched 600, 900, and 1800 cycles showed similar levels of message depression. Four hours following the initiation of stretch, message levels returning to nonstrained levels in all groups. We conclude that alterations in cell response to strain are in part mediated by gene expression, that alterations last 3–4 h in this system, and that the message mechanism itself exhibits a triggerresponse dependency to cycle number.

Use of the polymerase chain reaction to clone the potato leafroll virus coat protein gene directly from the total RNA of infected plants

The potato leafroll virus (PLRV) coat protein (CP) gene was directly cloned from the total RNA extracted from virus-infected plants. First strand cDNA synthesis was not necessarily specific; it was equally efficient using either random or CP-specific primers. The viral sequence encoding the coat protein was specifically amplified from the total population of cDNA molecules by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was cloned blunt-end into the pT7T318U plasmid vector, and the authenticity of the cloned gene verified by sequence analysis. This cloning strategy obviates the need for virus purification. Sequence comparison of the CP gene of the Italian isolate and those of five other PLRV isolates revealed a close similarity to the three European and the Canadian isolates, and a more distant relationship with the Australian one.

Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. Role of the autoinhibitory domain.

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca(2+)-dependent activation of its phosphatase activity. Substitution of Arg476 and Arg477 or Asp467 to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca(2+)-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A420 subunits alone have low Vmax and high Km (115 microM) values even in the presence of Ca2+. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca2+ binding to CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulates enzyme activity by lowering the Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca2+ acting through both CaM and the B subunit.

Molecular Cloning and Complete Nucleotide Sequence of Rice Ragged Stunt Oryzavirus Genome Segment 10.

cDNAs to rice ragged stunt Oryzavirus genome segment 10 (S10) were made by oligo-dT primed 1st strand cDNA synthesis after poly A addition to 3' terminal of viral transcripts and cloned in a bacterial plasmid pBR322. The overlapping cDNA clones were sequenced and the assembled sequence covered the full-length of the genome except for 22 nucleotides from the 5' terminus, which were determined by a direct RNA sequencing method. RRSV S10 is 1162 nucleotide long and an inverted repeat structure containing 6 nucleotides was found in the terminal region. It has a mini ORF in addition to the down stream long ORF. The upstream mini ORF encodes 12 amino acids. Presence of this mini ORF was confirmed by sequencing another full-length cDNA clones generated by reverse transcription coupled with a polymerase chain reaction. The long ORF encodes a polypeptide of 297 amino acids (Mr 32.4K).

Gonadotropin-releasing hormone (GnRH) and cyclic amp positively regulate inhibin subunit messenger RNA levels in human placental cells

Bioactive and immunodetectable levels of both inhibin and activin are present in the placenta, raising questions as to the regulatory control of their synthesis. This study was designed to determine the effect of cyclic AMP (cAMP) and gonadotropin-releasing hormone (GnRH) on inhibin subunit gene expression in short-term incubations of placental cells. A semi-quantitative polymerase chain reaction (PCR) technique was used after isolation of total RNA and first strand cDNA synthesis from mechanically dispersed trophoblast-enriched cells obtained from human placentae at term. The level of gene expression of inhibin subunits was higher for βA and α-subunits mRNA compared to the βB-subunit mRNA as determined by PCR in combination with Southern blotting or Northern hybridization. Steady-state levels of β-actin mRNA did not change throughout the 6-h incubation period and was used as a control of PCR amplification of the respective inhibin subunit gene transcripts following treatments with 8-bromo cAMP or GnRH. 8-bromo cAMP dose-dependently increased all three inhibin subunit gene transcripts with maximal responses seen at 150 μM (α-subunit mRNA 2.3-fold, βA-subunit mRNA 1.8-fold and βB-subunit mRNA 2.8-fold over control). GnRH (100 nM) significantly increased inhibin α and βB-subunit mRNA levels 2.9-fold and 2.0-fold, respectively ( P<0.01), but not βA-subunit mRNA. Collectively, the present findings demonstrate that in human term placental cells, gene expression of all inhibin subunits is under the direct influence of cAMP and further support a modulatory role of local GnRH in placental trophoblasts during late pregnancy.

Evolutionarily conserved elements in the 5' untranslated region of beta globin mRNA mediate site-specific priming of a unique hairpin structure during cDNA synthesis.

Generation of double-stranded cDNA during reverse transcription of a variety of mRNA molecules is well known to involve the formation of covalently linked antisense and sense strands in a hairpin configuration. In the present study we have examined the sequence of molecular events which occurs during cDNA synthesis from mouse beta globin mRNA, in particular the self-priming event that initiates synthesis of sense-strand DNA. Upon completion of reverse transcription of globin mRNA and the removal of RNA template by RNase H activity associated with reverse transcriptase, the 3' end of cDNA snaps back to form a stable double-stranded structure, which is extended by reverse transcriptase to generate the sense DNA strand. Surprisingly, the fourteen 3' terminal nucleotides of the beta globin antisense DNA strand (cDNA) have strong complementarity with an internal segment of the same molecule corresponding to a portion of the 5'-untranslated region of the mRNA located just upstream of the translation start site. Efficient second strand cDNA synthesis appears to require the occurrence within the cDNA molecule of these two complementary elements, one of which must be 3'-terminal. A second surprising feature is that the strong complementarity between the terminal and the internal portions of the molecule exists in the antisense DNA and not in the sense mRNA strand. This is because A:C mismatches on the sense strand correspond to relatively stable T:G base pairs on the antisense strand. Such an extended region of complementarity within the segment of cDNA corresponding to the short 5' untranslated region of beta globin mRNA is unlikely to occur purely by chance, suggesting some underlying function. In this regard it is of interest that cDNAs of adult beta globin mRNAs from other mammalian species show a very similar arrangement of complementary elements, and that complementarity is heavily conserved, even when there are substitutions in nucleotide sequence.

SEMIQUANTITATIVE MEASUREMENT OF GENE-EXPRESSION BY RT-PCR - A CAUTIONARY TALE

The polymerase chain reaction (PCR) theoretically offers a very powerful means of quantifying gene expression in cells and tissues of different histological classes. From a technical viewpoint however, the use of RT-PCR to quantify gene expression can be demanding with poor reproducibility arising from several diverse sources. In this study, we describe and fully characterise an RT-PCR assay which generates highly reproducible estimates of DNA polymerase beta gene expression relative to the expression of beta-actin in a semi-quantitative manner. Particular emphasis has been placed on the efficiency of first strand cDNA synthesis and to aspects of primer design. In addition, various aspects associated with the quantification of gene expression required to generate reproducible results are discussed. Using the techniques described herein, the quantification of DNA polymerase beta expression in three independent experiments using human colon carcinoma cells was highly reproducible with ratios of target to internal standard gene expression of 3.68x10(-4) +/- 0.23x10(-4). Provided that careful consideration is given to key areas of the RT-PCR assay during experimental work up procedures, this assay can be used to provide an accurate measure of gene expression in cell lines.

Dahl's salt-resistant normotensive rat has mutations in cytochrome P450(11 beta), but the salt-sensitive hypertensive rat does not.

Molecular cloning of cytochrome P450(11 beta) cDNAs from the adrenal glands of Dahl's salt-sensitive hypertensive (DS) and salt-resistant normotensive (DR) rats was performed using a combined technique of the first strand cDNA synthesis by reverse transcriptase followed by polymerase chain reaction. The cDNA sequence of P450(11 beta)-DS was identical to that of wild type P450(11 beta). In contrast, the clone obtained from the DR rat contained six nucleotide substitutions causing five amino acid alterations (Arg-127-->Cys, Val-351-->Ala, Val-381-->Leu, Ile-384-->Leu, and Val-443-->Met). When the two cDNAs were expressed in COS-7 cells and steroid conversion rates of the transformed cells were determined, a ratio of 18-hydroxylation to 11 beta-hydroxylation of 11-deoxycorticosterone by P450(11 beta)-DS-expressed cells was 0.58, whereas that by P450(11 beta)-DR-expressed cells was 0.23. Plasma levels of 18-hydroxy-11-deoxycorticosterone and corticosterone (the 11 beta-hydroxylation product of 11-deoxycorticosterone) in DS and DR rats well reflected the steroidogenic activities of the two P450s. These results suggest that the characteristic plasma steroid level of the DR rat is caused by the mutations in P450(11 beta) gene and may act to maintain the normotensive blood pressure in this rat strain during sodium loading.

Arbitrary primed PCR fingerprinting of RNA applied to mapping differentially expressed genes.

Differential gene expression between various tissues and developmental stages or between cells in vitro under different growth conditions can be rapidly and efficiently compared using the RNA arbitrarily primed polymerase chain reaction (RAP) fingerprinting method (Welsh et al., 1992b; Liang and Pardee, 1992). In RAP, a primer of arbitrary sequence primes both first and second strand cDNA synthesis. The mixture of products is then PCR amplified and resolved electrophoretically, yielding highly reproducible fingerprints that are tissue-specific or growth condition-specific. Differences between fingerprints arise from differentially expressed genes, as verified by Northern blot analysis. RAP can be performed on the RNA samples using various DNA primers. Each two day experiment yields a sample of approximately twenty cDNA products per lane making the identification of differentially or developmentally regulated genes no longer rate limiting. Those PCR products representing genes that are regulated can be cloned from the gel and sequenced. Sequences can be compared to the DNA and protein sequence databases to identify homologs, motifs and members of gene families. The clones can be placed on the genetic map as Expression Tagged Sites (ETS, Adams et al., 1991a).

Molecular Characterization of the Soybean Alcohol Dehydrogenase Gene Family Amplified in Vitro by the Polymerase Chain Reaction

Soybean (Glycine max) alcohol dehydrogenase (ADH) cDNAs were amplified in vitro from total RNA by the polymerase chain reaction (PCR). The amplification strategy involved first strand cDNA synthesis from anaerobic cotyledon total RNA using an 18-thymidine primer. The second strand cDNA primer was a conserved sequence near the 5' end of known plant ADH transcripts. The PCR products were ligated into a plasmid vector and unique clones were isolated on the basis of size and restriction pattern. Sequence analysis revealed three distinct classes of soybean ADH cDNAs, all of which showed high homology to Adh genes from maize and peas. RNA blot hybridization analyses showed differential expression patterns for these genes. One gene, expressed constitutively in all seedling organs, was inducible by anaerobiosis, one gene was expressed only in anaerobic organs, and the third gene was expressed predominantly in anaerobic roots.

Cloning and sequencing of a jack bean urease-encoding cDNA

A cDNA which encodes the entire amino acid (aa) sequence of the mature jack bean urease has been cloned in Escherichia coli from a library prepared from the mRNA of developing jack beans. It was necessary to use reverse transcriptase in the second strand cDNA synthesis m order to obtain full-length cDNA. The complete cDNA was obtained in the form of two contiguous DNA fragments, each of which was completely sequenced. The conceptual translation of the nt sequence gave an 840-aa sequence which was identical to the directly determined sequence except for one conservative aa substitution (Takashima et al., Eur. J. Biochem. 175 (1988) 151–165). These data constitute the first report on the cloning and sequence of the cDNA encoding a urease from any higher plant.

A highly efficient directional cDNA cloning method utilizing an asymmetrically tailed linker-primer plasmid

A new procedure using an asymmetrically tailed linker-primer plasmid has been developed to prepare extremely high complexity cDNA libraries. This procedure yields plasmid primed libraries with a final form equivalent to those made by the procedure of Okayama and Berg. However, the number of steps involved in library preparation is decreased. The form of the vector is such that one end of the linearized linker-primer plasmid has a 3' terminal extension of 40 deoxythymidylate residues (the dT end). The other end has a 3' terminal extension of 10 deoxycytidylate residues (the dC end). The dC end of the plasmid is blocked to further 3' extension by a 3' phosphate group. This configuration enables one to prime first strand cDNA synthesis at the dT end, tail the 3' end of the cDNA with deoxyguanylate residues without tailing the dC end (due to the 3' phosphate block). The plasmid primed cDNA can then be self-annealed and the 3' phosphate blocking group removed during the synthesis of double stranded cDNA. The efficiency of this procedure is significantly higher than other methods (including phage based libraries): linker-primer libraries have 15 to 900-fold higher complexity than libraries prepared by other methods. A cloning efficiency of 9 x 10(8) colonies per microgram of linker-primer DNA was achieved. This method should be useful for the cloning of cDNAs corresponding to extremely rare mRNAs.

Amino acid substitutions Phe66—-Leu and Ser126—-Pro abolish cortisol and aldosterone synthesis by bovine cytochrome P450(11)beta

A cDNA clone encoding the complete protein sequence of the precursor form of bovine cytochrome P450(11)beta has been constructed using a combined technique of first strand cDNA synthesis by reverse transcription followed by polymerase chain reaction. Upon expression of this cDNA in COS 1 cells the P450(11)beta is found to be proteolytically processed and localized in the mitochondrion. This cDNA encodes the major form of P450(11)beta found in bovine adrenal cortex (designated 11 beta-3; Kirita, S., Morohashi, K., Hashimoto, T., Yoshioka, H., Fujii-Kuriyama, Y., and Omura, T. (1988) J. Biochem. 104, 683-686) and is capable of catalyzing 11 beta-hydroxylation of deoxycorticosterone, 11-deoxycortisol, and androstenedione in COS 1 cells as well as aldosterone synthesis from deoxycorticosterone. In addition, a second form of P450(11)beta (herein designated 11 beta-4), having no detectable 11 beta-hydroxylase activity or aldosterone synthase activity, was found in the local bovine population by this cloning procedure. These two forms of P450(11)beta (11 beta-3 and 11 beta-4) contain five amino acid differences between them, all located within the amino-terminal half of the molecules. By changing 2 of the amino acids in the inactive form to the corresponding amino acids in the active form (Leu66----Phe and Pro126----Ser) both 11 beta-hydroxylase and aldosterone synthetase activities were completely restored. Neither of these changes alone led to detectable activity. Thus, upon expression in mitochondria of heterologous cells, bovine P450(11)beta catalyzes both 11 beta-hydroxylation and aldosterone synthesis as reported previously for the purified enzyme in an in vitro reconstituted system, and Phe66 and Ser126 seem to be important residues in maintaining both activities.

Detection of point mutations in type I collagen by RNase digestion of RNA/RNA hybrids

We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the alpha 1(I) and alpha 2(I) chains of type I collagen. Point mutations are detected by RNase A cleavage of mismatches in RNA/RNA hybrids. The mRNAs coding for the fibrillar collagens present special problems for hybrid analysis because of their large size and their GC-rich and repetitive sequences. We have generated a series of overlapping antisense riboprobes covering the entire pro alpha 1(I) and pro alpha 2(I) mRNAs. Uniformly labelled normal antisense riboprobes are hybridized with the total fibroblast RNA of patients with possible mutations in type I collagen. Mismatches in the resulting RNA/RNA hybrids are cleaved with RNase A and the labelled riboprobe cleavage products are examined electrophoretically. The sensitivity and specificity of the system were demonstrated by the detection and localization of a known point mutation in the codon for alpha 1(I) glycine 988 (1). DNA for sequencing the mutations localized by hybrid analysis may be obtained by either (1) generation of a fibroblast cDNA library and isolation of both alleles by plaque screening, or (2) a more rapid method using first strand cDNA synthesis from poly (A+)-mRNA, followed by PCR amplification of the mutation-containing region of the DNA/RNA hybrid. This strategy for detection and isolation has wide application not only for mutations causing connective tissue disorders, but also for mutations in other large and repetitive genes. We have used this strategy for the detection and sequencing of a point mutation in alpha 2(I) mRNA associated with a case of lethal osteogenesis imperfecta. The G----A point mutation in the codon for alpha 2(I) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.

Conservation of the 3′ terminal nucleotide sequence in five carlaviruses

Carnation latent virus (CLV) is the type member of the carlavirus group (1), a group with positive sense RNA genomes of Mr 2.3-3.0X 10 which are polyadenylated at the 3' terminus. The nucleotide sequence of the 3' proximal regions of four carlaviruses: potato virus M (PVM), potato virus S (PVS), lily symptomless virus (LSV) and Helenium virus S (HelVS) have recently been presented (2, 3, 4, 5). Using a similar procedure for cloning, employing oligo (dT) priming of first strand cDNA synthesis, we have generated a library of 3' terminal clones of CLV RNA which have been sequenced by the dideoxynucleotide termination procedure. An examination of the 3' sequence of PVM (2), PVS (3), LSV (4) and HelVS (5) RNAs and a comparison with the 3' nucleotide sequence of CLV RNA reveals some striking similarities. These similarities are conserved in all five viruses in the central region of a putative 3 ' terminal openreading frame (ORF) coding for polypeptides of 12.6, 10.8, 11.6, 10.7 and 16 KDa for respectively HelVS, PVM, CLV, PVS and LSV (Figure 1). Within this region of high conservation four Cys residues which conform to a consensus sequence for a putative 'zinc-finger' nucleic acid-binding domain (6) are also conserved. All five proteins contain a high proportion of positively charged residues with CLV containing 15 Arg and 9 Lys residues and 10 residues of Glu and Asp. This 3' terminal ORF appears to be unique to carlaviruses and sets them apart from other virus groups but no direct evidence for its expression in vivo or in vitro has been presented (2, 3, 4, 5). Analysis of the 3' terminal non-coding regions of the five RNAs using the UWGCG GAP program (7) revealed over 60% homology between the nucleotide sequences of CLV compared with the other four viruses (Figure 2). This 3' region of the viral RNAs has the capacity to be folded into a series of stem-loop structures. Such strict sequence conservation at the 3' terminus of carlaviruses may have implications with regard to RNA secondary structure and configuration and might be important in RNA replication. However the putative polyadenylation signal AATAAA present in the terminal sequence of HelVS (5) and PVM RNAs (2) is not conserved in all of the viruses.

Rapid amplification of complementary DNA from small amounts of unfractionated RNA

We have combined, in a rapid and straightforward manner, the synthesis and subsequent amplification of individual cDNA sequences from microgram quantities of unfractionated total RNA. Taq1 polymerase, a thermostable DNA polymerase, and Moloney murine leukemia virus (MMLV) reverse transcriptase share similar buffer conditions; these reactions can be performed sequentially, in a single tube, without the need for purification or changes of buffer after the synthesis of cDNA. In this way, nonspecific losses of material are minimized and the required number of cells is reduced. Cell numbers, particularly from human tissues, can be limiting; the requirement for only small amounts of unfractionated RNA makes possible the isolation and characterization of cDNAs from biological materials available in limited quantities. As a demonstration system, we report the rapid synthesis and amplification of cDNA sequences corresponding to the first exon of human immunoglobulin E (IgE). MMLV reverse transcriptase is used with specific (i.e., IgE) or generic (i.e., oligo-[dT(12–18]) oligomers to prime first strand cDNA synthesis from unfractionated RNA isolated from a human myeloma line, U-266. The necessary primers, deoxynucleotides and Taq1 polymerase, required for second strand cDNA synthesis and the subsequent logarithmic amplification process, are then added to the reaction mixture. This technique provides a useful means of characterizing expressed and processed gene transcripts.

Leukocyte Adhesion Deficiency: Aberrant splicing of a conserved integrin sequence causes a moderate deficiency phenotype

Leukocyte adhesion deficiency (LAD) is a heritable deficiency of the LFA-1, Mac-1, p150,95 family of leukocyte alpha beta heterodimers (the leukocyte integrins). We have studied the defect in patients who synthesize an aberrantly small form of the beta subunit common to all three proteins. S1 nuclease protection showed the presence of a 90-nucleotide mismatch in RNA from patients and relatives, correlating with inheritance of the disease. Use of the Taq polymerase chain reaction to amplify this region of RNA after first strand cDNA synthesis and sequencing showed an in-frame deletion of 90 nucleotides in the extracellular domain. Thus, this highly conserved region, 63% and 53% identical in amino acid sequence to two other beta subunits of the integrin family, is required for association of the beta subunit with alpha subunits. The 90-nucleotide region corresponds to a single exon present in both the normal and patient genome. The patient DNA has a single G to C substitution in the 5' splice site. This results in the direct joining of nonconsecutive exons in an unusual type of abnormal RNA splicing. A small amount of normally spliced message, detected by S1 nuclease protection and Taq polymerase chain reaction, encodes a normal sized beta subunit which is surface-expressed and accounts for the low levels of leukocyte integrin expression observed in these patients, and hence the moderate phenotype.

Sindbis virus mutant ts20 of complementation group E contains a lesion in glycoprotein E2

A technique has been devised to readily obtain the entire structural protein region of Sindbis virus cloned into a plasmid vector. This method uses the fact that the nearest site for restriction enzyme HindIII to the 3′ terminal poly(A) occurs at nucleotides 6266–6271 in the genomic RNA. Inserts extending from the poly(A) tract to this HindIII site are 5438 nucleotides long (excluding the poly A tract) and contain the entire 4106-nucleotide structural protein region. Using an oligo(dT)-tailed vector as a primer for first strand cDNA synthesis such clones could be obtained in high yield. We were interested in a precise determination of the mutation responsible for the temperature-sensitive phenotype of ts20, a mutant belonging to complementation group E which has a defect in the function of glycoprotein E2 at the nonpermissive temperature. Using this technique we have cloned and sequenced the structural protein region of ts20 and of several revertants and concluded that the mutation was a change from histidine to leucine at amino acid 291 of E2. Reversion to temperature insensitivity occurred by same site reversion to the parental nucleotide, restoring the original histidine as amino acid 291. Thus, complementation group E of Sindbis virus results from changes in glycoprotein E2 and together with previous results from our laboratory ( Arias et al., 1983 ; Hahn et al, 1985 ) demonstrates that the three RNA + complementation groups of Sindbis virus, C, D, and E, result from changes in the three structural proteins of the virus, capsid, glycoprotein El, and glycoprotein E2, respectively