Intricate combinatorial patterns of exon splicing generate multiple Rh-related isoforms in human erythroid cells

The Rhesus (Rh) blood group system shows complex polymorphisms in the human. Some of the heterogeneity may be generated by alternative RNA splicing. For a systematic analysis of Rh-related mRNA isoforms expressed in reticulocytes, we isolated mRNA, which was then reverse transcribed and amplified by the polymerase chain reaction (PCR) to give Rh-related cDNAs of two segments of 704 bp and 975 bp. The PCR amplification of the 5'-region yielded a single PCR product, whereas a complex electrophoretic pattern of PCR bands was derived from the 3'-region. A highly reproducible ladder of multiple additional bands migrated below the PCR products corresponding to the full-size cDNAs for RhPI and RhPII and encoding two different Rh polypeptides. Eleven and five truncated isoforms of the RhPI and RhPII cDNAs, respectively, were identified in the PCR products. These isoforms appear to be generated by combinatorial splicing of six RhPI and three RhPII exons. Our results suggest that the Rh-related polypeptides consist of a mixture of RhPI and RhPII polypeptide isoforms differing at the C terminus. Multiple RNA splicing pathways are thus operative in the two Rh-related genes even within a single cell lineage of human erythroid cells.     

Determination of the sequence of the arylacetyl acyl-CoA:amino acid N-acyltransferase from bovine liver mitochondria and its homology to the aralkyl acyl-CoA:amino acid N-acyltransferase

The arylacetyl acyl-CoA:amino acid N-acyltransferase was previously purified to homogeneity from bovine liver mitochondria, and partial sequences were obtained for peptides generated by cyanogen bromide cleavage of the enzyme. One of these sequences was used to design an oligonucleotide probe that was utilized to screen a bovine liver cDNA library. Several clones were isolated and sequenced, and the sequence is given. The cDNA contains 346 bases of 5'-untranslated region and 439 bases of 3' untranslated region. The cDNA codes for an enzyme containing 295 amino acid residues. The sequence gives a molecular weight for the enzyme of 38,937, which is larger than that previously estimated for the functional enzyme, which suggests the existence of ca. 5 kDA of signal peptide. The molecular weight of the enzyme was slightly lower than that of the aralkyltransferase, which was previously determined to be 39,229. Comparison of this sequence with that which we previously obtained for the aralkyltransferase indicated that the coding regions were of identical length and that the sequences were 78% homologous. However, the 5' and 3' untranslated regions had less than 29% homology. The derived amino acid sequences were 71% homologous. This high homology indicates a common origin for the two enzymes. There are, however, significant differences in amino acid compositions, and these are discussed.     

Giant catfish Pangasianodon gigas growth hormone-encoding cDNA: cloning and sequencing by one-sided polymerase chain reaction

cDNA clones encoding giant catfish (Pangasianodon gigas) growth hormone (GH) have been isolated using a polymerase chain reaction (PCR) strategy. Pairwise combinations of degenerate and general primers allowed for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products were cloned and sequenced. The cDNA sequence was found to encode a polypeptide of 200 amino acids (aa), including a putative signal peptide of 22 aa. The 5' and 3' untranslated regions of the message are 58 and 515 nucleotides long, respectively. The giant catfish GH displays the highest aa sequence homology with the carp GH, with 80% of sequence identity. Moreover, giant catfish GH has structural features in common with both mammalian and avian GH polypeptides, and also contains the domains of conserved sequence found in other GH.     

TCDD reduces rat hepatic epidermal growth factor receptor: comparison of binding, immunodetection, and autophosphorylation

Two overlapping cDNA clones for core protein of a biglycan of bovine aorta were isolated from a pSPORT bovine aorta tissue cDNA library. The 2043-bp cDNA contains a 114-bp 5' untranslated region, a 1224-bp cDNA open reading frame and a 705-bp 3' untranslated region. The encoded core preproprotein contains a prepeptide (residues no. -37 to -19) and a propeptide (residues no. -18 to -1), with 369 amino acid residues corresponding to a molecular mass of 41.6 kDa. The deduced amino acid sequence revealed a striking homology to rat vascular smooth muscle cell, human bone and bovine articular cartilage biglycans from cell culture.     

RNA binding properties of core protein of the flavivirus Kunjin

Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed in E. coli. They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5' untranslated region (5' UTR, 93 to 96 nucleotides), the 3' UTR (624 nucleotides), and the 5' UTR plus most of the C coding region (5' core, 440 nucleotides). Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA. In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxy-terminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA. These small domains probably acted co-operatively in binding of mature C to KUN RNA probes. The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.     

Morphometric variables of developing primary maxillary first molar crowns in humans

Complementary DNA sequences were selected from a resource of tentatively identified clones from a porcine small intestine cDNA library. Forty PCR primer pairs were designed to amplify 101-309 base pairs of the 3' untranslated region of the genes. The PCR conditions were optimized by altering both formamide and magnesium concentrations on samples of pig, mouse, and hamster DNA. Twenty primer pairs that, under stringent conditions, were pig-specific and amplified the expected fragments were chosen for regional assignment in a pig/rodent hybrid cell panel. Furthermore, 22 primer pairs were chosen to amplify DNA from the parental animals of the PiGMaP shared reference families in order to detect possible polymorphisms. Primer pairs that generated polymorphisms were used for genetic mapping. A total of 22 porcine expressed sequence tags (ESTs) were cytogenetically or genetically mapped by this approach. Twelve of the mapped ESTs could be added to the human-porcine comparative map.     

Alternative splicing of a Caenorhabditis elegans gene produces two novel inhibitory amino acid receptor subunits with identical ligand binding domains but different ion channels

Two full-length cDNAs, gbr-2A and gbr-2B, encoding inhibitory amino acid receptor subunits have been amplified and cloned from Caenorhabditis elegans mRNA. The 5' 732 bp of the two cDNAs, encoding 237 amino acids, are identical. The 3' 758 bp of the gbr-2B cDNA are present within the 3' untranslated region of the gbr-2A clone. As a result, the two cDNAs are predicted to encode subunits which share a common extracellular N-terminal sequence of 237 amino acids, but different, though closely related, C-terminal sequences which include four predicted membrane-spanning regions. A search of the EMBL database revealed that the sequences of the two subunits are most closely related to the alpha-subunit of the C. elegans avermectin receptor. Northern blot analysis showed the presence of two related mRNAs of approximately 2.2 and 1.5 kb in a developmentally mixed population of C. elegans. The genomic DNA sequence confirms that both mRNAs were transcribed from the same gene, gbr-2, suggesting that the closely related 3' sequences have arisen as a result of a partial gene duplication event. We propose that C. elegans is utilising alternative splicing to generate receptor subunits with identical extracellular, ligand-binding domains but different transmembrane, channel forming domains.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in 12 New York hospitals. MRSA Collaborative Study Group

Hepatitis C virus (HCV) sequences recovered from serum, peripheral blood mononuclear cells (PBMCs), and various tissues from human immunodeficiency virus type 1 (HIV-1) positive patients were compared by single strand conformational polymorphism (SSCP) and sequencing. In five patients, paired serum and PBMCs samples were analyzed while in two other patients multiple autopsy tissues were studied. Sequences amplified from the NS5 and E2 regions were consistently identical in the same patient; however, three PBMCs samples and three different tissue samples (pancreas and adrenal gland in one patient and lymph node in the other patient) contained 5' untranslated region (5'UTR) sequences that were different from circulating sequences. The presence of 5' UTR sequences differing from circulating sequences correlated with the presence of HCV RNA negative strand, as the latter was detected by a Tth-based strand-specific assay in all but one of these samples. These two independent lines of evidence: viral sequence differences and the presence of RNA negative strand in the same tissues strongly argue for the genuine presence of extrahepatic HCV replication, at least in the setting of HIV-1 infection.     

Adult specific expression and induction of cytochrome P450lpr in house flies

Arabinogalactan-proteins (AGPs) were isolated from the pistils of Nicotiana alata, deglycosylated, and the protein backbones fractionated by reversed-phase HPLC as previously reported. A major fraction, RT35 was isolated and peptide sequences were obtained after protease digestion. A gene-specific degenerate oligonucleotide was designed according to the amino acid sequences and a 380 bp PCR fragment was amplified in vitro from pistil RNA. The PCR fragment was used to screen a pistil cDNA library and a 762 bp cDNA clone (AGPNa3) was isolated and sequenced. The AGPNa3 cDNA encodes a 169 amino acid protein which consists of three domains: an N-terminal secretion signal, a Pro-rich domain and a C-terminal Cys-rich domain. The mature protein has 145 amino acid residues (16.7 kDa) and a predicted pl of 7.5. Northern blot analyses showed that the AGPNa3 gene was only expressed in the pistils of N. alata and of closely related Nicotiana species but not in other plants or suspension-cultured cells. Further Northern blot analysis and in situ hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.     

A catalogue of genes in the cardiovascular system as identified by expressed sequence tags

The heart, which is composed of all the cellular components of the circulatory system, is a representative organ for obtaining genes expressed in the cardiovascular system in normal and disease states. We used partial sequences of cDNA clones, or expressed sequence tags, to identify and tag genes expressed in this organ. More than 3500 partial sequences representing > 3000 cDNA clones have been obtained from either the 5' or 3' end of inserts derived from human heart cDNA libraries. Of 3132 cDNA clones analyzed by sequence similarity searching against the GenBank/EMBL data bases, 1485 (47.4%) were found to represent additional, previously undiscovered genes, whereas 267 clones were matched to human brain expressed sequence tags. Clones matching to known genes were catalogued according to their putative structural and cellular functions. cDNA probes from reverse-transcribed mRNAs of fetal and adult hearts were used to study differential expression of selected clones in cardiac development. Cataloguing genes expressed in the heart may provide insight into the genes involved in health and cardiovascular disease.     

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.     

PCR-mediated recombination: a general method applied to construct chimeric infectious molecular clones of plasma-derived HIV-1 RNA

A PCR-based approach was developed that provides a powerful tool for engineering recombinant molecules without reliance on restriction sites. DNA sequences were first amplified by high-fidelity PCR using Pfu polymerase; they were then used both as 'megaprimers' and templates in subsequent asymmetric long PCR amplifications to form chimeric clones. To demonstrate the technique, we constructed chimeric full-length HIV-1 clones derived from reverse-transcribed plasma viral RNA and proviral LTRs. Biologic characterization of these clones showed that most were infectious in tissue culture and sequence analysis demonstrated an error rate of only one base change in 20 kb of DNA sequence. For PCR-mediated recombination, it is necessary to know the sequence of the 3' and 5' overlapping regions of the desired PCR products. This method may be extended to include construction of chimeras between any DNA fragments lacking sequence homology. Such chimeras may be constructed by introducing overlapping sequences to one of the fragments. To ensure that unwanted mutations have not been introduced into the clones constructed by this method, each clone should be sequenced. Our results demonstrate that by using a high-fidelity polymerase and highly controlled PCR conditions, the PCR-introduced error rate can be greatly minimized. This new procedure may be used to construct infectious chimeras of HIV or SIV for studies of vaccines and pathogenesis. Moreover, the method is designed to exchange viral genes at precise boundaries to study individual gene products from different HIV genomes. It can also be used to construct expression vectors for production of specific proteins or delivery vectors for gene transfer and gene therapy. Finally, the technique described here provides a versatile tool to transfer genes or gene fragments from different sources for genetic investigation and engineering.