Showing papers on "genomic DNA published in 1987"
TL;DR: It is suggested that a co- or posttranscriptional C----U change may result in the production of apo-B48, which represents the amino-terminal 2152 amino acids of api-B100, which is the first example of tissue-specific modification of a single mRNA nucleotide resulting in two different proteins from the same primary transcript.
966 citations
TL;DR: The development of two new methods for detecting and localizing single base changes in cloned and genomic DNA are described, likely that the use of both methods will be complementary, resulting in the detection of a very large fraction of all possible substitutions.
Abstract: Publisher Summary This chapter discusses the detection and localization of single base changes by denaturing gradient gel electrophoresis For most purposes, single-stranded deoxyribonucleic acid (DNA) probes 140–1000 bases in length are used, which allows about half of this number of base pairs to be screened for base changes in a single gel lane The chapter discusses the use of single-stranded RNA probes It describes the development of two new methods for detecting and localizing single base changes in cloned and genomic DNA In both procedures, a single-stranded, radioactively labeled probe of wild-type sequence is annealed to cloned or genomic DNA If the DNA under test carries a single base change, a hybrid double-stranded species containing a mismatch is formed In the first procedure, either a ribonucleic acid (RNA) or a DNA probe is used, and the mismatched duplex is separated from the perfectly paired, wild type duplex by electrophoresis in a denaturing gradient gel In the second procedure, the probe is composed of single-stranded RNA and the mismatch is cleaved by ribonuclease; the resulting cleaved products are examined by polyacrylamide gel electrophoresis and autoradiography It is estimated that each method will detect approximately 50% of all possible substitutions, insertions, or deletions in a probed region Although there is some overlap in the base changes that are detected with the two procedures, it is likely that the use of both methods will be complementary, resulting in the detection of a very large fraction of all possible substitutions and they should be particularly useful in mapping and diagnosing small mutations that result in genetic disease This method has led to the detection of neutral polymorphisms for genetic linkage studies The chapter lists all the equipment needed to prepare and run the gels and also describes the hybridization procedure for RNA probes
938 citations
TL;DR: Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel, the A channel of Drosophila melanogaster.
Abstract: On the basis of electrophysiological analysis of Shaker mutants, the Shaker locus of Drosophila melanogaster has been proposed to encode a structural component of a voltage-dependent potassium channel, the A channel. Unlike sodium channels, acetylcholine receptors, and calcium channels, K+ channels have not been purified biochemically. To facilitate biochemical studies of a K+ channel, genomic DNA from the Shaker locus has been cloned. Rearrangements in five Shaker mutants have been mapped to a 60-kilobase segment of the genome. Four complementary DNA clones have been analyzed. These clones indicate that the Shaker gene contains multiple exons distributed over at least 65 kilobases of genomic DNA in the region where the mutations mapped. Furthermore, the gene may produce several classes of alternatively spliced transcripts. Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel.
725 citations
TL;DR: Yeast transcriptional activators encoded by E. coli genomic DNA fragments fused to the coding sequence of the DNA-binding portion of GAL4 and a fusion protein that contains no yeast protein sequence but activates transcription in yeast are described.
667 citations
TL;DR: A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gamblingiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns.
Abstract: A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3' end of the 28S beta coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.
591 citations
TL;DR: The role of A X T/G X C pressure in diversification of bacterial DNA sequences and codon usage patterns is discussed in the perspective of the neutral theory of molecular evolution.
Abstract: The genomic guanine and cytosine (G + C) content of eubacteria is related to their phylogeny. The G + C content of various parts of the genome (protein genes, stable RNA genes, and spacers) reveals a positive linear correlation with the G + C content of their genomic DNA. However, the plotted correlation slopes differ among various parts of the genome or among the first, second, and third positions of the codons depending on their functional importance. Facts suggest that biased mutation pressure, called A X T/G X C pressure, has affected whole DNA during evolution so as to determine the genomic G + C content in a given bacterium. The role of A X T/G X C pressure in diversification of bacterial DNA sequences and codon usage patterns is discussed in the perspective of the neutral theory of molecular evolution.
571 citations
TL;DR: This work studied the genomic DNA of five patients with β-thalassaemia, and found two previously undescribed mutations, along with three known alleles, including the first natural mutation observed at the cap site of the β-globin gene.
Abstract: Direct sequencing of specific regions of genomic DNA1 became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA2–5. Recently, human mitochondria! DNA was amplified and directly sequenced6. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R. K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with β-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106–107 and the other is an A–C transversion at the cap site (+1) of the β-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.
509 citations
TL;DR: The topological specificity in the transcription of Dl corresponds to the one expected for a regulatory signal that mediates epidermal commitment, and transcripts homologous to Dl are detected in all territories with neurogenic abilities, e.g. the neurogenic ectoderm and the primordia of the sensory organs.
Abstract: The decision of an ectodermal cell to take on a neural or an epidermal fate depends on its interactions with the neighbouring cells. In Drosophila melanogaster, the available evidence suggests that a regulatory signal necessary for epidermal commitment is built by the products of the so-called neurogenic genes. We have cloned 180 kb of genomic DNA surrounding the neurogenic gene Delta (Dl). Restriction fragment-length polymorphisms were mapped to a region of 25 kb. These 25 kb of DNA are assumed to contain essential parts, or all, of the Dl gene. Northern blots detect two developmentally regulated transcripts, of 5.4 and 4.6 kb, which are associated with the region where the mutants map. Serveral cDNA clones were recovered from embryonic cDNA libraries by homology to the 25 kb of genomic DNA. The complete sequence of a cDNA clone containing an insert of 4.73 kb was determined. The conceptual translation of the longest open reading frame yields a protein of 880 amino acids. This protein displays characteristics of a membrane protein, with intercellular, transmembrane and extracellular domains. The extracellular domain contains a tandem array of nine EGF-like repeats. In in situ hybridizations to tissue sections, transcripts homologous to Dl are detected in all territories with neurogenic abilities, e.g. the neurogenic ectoderm and the primordia of the sensory organs. Initially all cells of these neurogenic territories express Dl, but later on transcription of Dl becomes restricted to the cells that have adopted the neural fate. The topological specificity in the transcription of Dl corresponds to the one expected for a regulatory signal that mediates epidermal commitment.
326 citations
TL;DR: A comparison of the human and mouse LCA protein sequences revealed a marked difference only in the extracellular domain, and Northern blot analysis with LCA exon-specific probes demonstrates the existence of at least two more LCA mRNA forms that are generated by differential splicing.
Abstract: Leukocyte common antigens (LCAs, also known as T200 and CD 45) are integral membrane proteins expressed exclusively on hematopoietic cells. These molecules exhibit varying molecular masses and epitopes when expressed in different cell types. To determine the genetic bases for the generation of this diversity, three classes of human LCA cDNA clones that are different near their 5' ends have been isolated. These differences arose as a result of differential usage of three exons as determined from an analysis of a genomic DNA clone. Furthermore, Northern blot analysis with LCA exon-specific probes demonstrates the existence of at least two more LCA mRNA forms that are generated by differential splicing. A comparison of the human and mouse LCA protein sequences revealed a marked difference only in the extracellular domain.
311 citations
TL;DR: The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2/0 mouse myeloma cells resulted in the production of functional IgG that retained the specific binding to the surface antigen 17-1A expressed on colorectal carcinoma cells.
Abstract: We have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and we have inserted them into mammalian expression vectors containing genomic DNA segments encoding human gamma 3 and kappa constant regions The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2/0 mouse myeloma cells resulted in the production of functional IgG that retained the specific binding to the surface antigen 17-1A expressed on colorectal carcinoma cells
TL;DR: This study has used PCR/SSO to analyze partially purified DNA extracted from formalin-fixed, paraffin-embedded tissue specimens, and reports that this DNA, including samples that were partially degraded, proved to be suitable for analysis by the PCR/ SSO procedure.
TL;DR: Analysis of the PDR1 deduced amino acid sequence revealed several homologies to four different regulatory proteins involved in the control of gene expression, including a cysteine-rich motif suggested to be a metal-binding domain for DNA recognition.
TL;DR: Southern blot analysis of human genomic DNA digested with different restriction enzymes strongly suggests the existence of a multigene family for cyclophilin.
Abstract: Complementary DNA encoding human cyclophilin, a specific cyclosporin A-binding protein, has been isolated from the leukemic T-cell line Jurkat and sequenced. Comparison of the deduced amino acid sequence with the previously determined sequence of bovine thymus cyclophilin reveals only three differences: an additional amino acid at the carboxy terminus end and two internal changes. RNA transfer blot analysis indicates an mRNA size of approximately 1 kb for human T-cell cyclophilin. Phytohaemagglutinin and phorbol myristate acetate induction of T cells treated or not with cyclosporin A affects only marginally the level of cyclophilin mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes strongly suggests the existence of a multigene family for cyclophilin.
TL;DR: In this article, a novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus, which was selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation.
Abstract: A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.
TL;DR: The size, distribution, and sequence homology of the introns within the gene were compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases, and the gene for human prothrombin was screened for positive lambda phage.
Abstract: A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were the compared to those ofmore » the genes for the other vitamin K dependent proteins and several other serine proteases.« less
TL;DR: Six different classes are found based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA.
Abstract: Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.
TL;DR: Cosmid vectors for genomic walking and restriction mapping have been proposed in this paper, which contain the transcription promoters from either bacteriophage SP6, T7, or T3 flanking a unique BamHI cloning site.
Abstract: We have designed cosmid vectors for rapid genomic "walking" and restriction mapping. These vectors contain the transcription promoters from either bacteriophage SP6, T7, or T3 flanking a unique BamHI cloning site. Mammalian expression modules encoding the dominant marker neomycin phosphotransferase or the amplifiable dihydrofolate reductase gene expressed from SV40 promoters were inserted for use in gene transfer studies. Restriction sites for the enzymes Not I and Sfi I, which cut mammalian DNA very infrequently, have been engineered near the transcriptional promoters to enable the excision of most inserts as single, full-length fragments. Genomic libraries representative of mouse, human, and hamster genomes were constructed by inserting 33- to 44-kilobase-pair (kbp) DNA fragments, generated by partial cleavage of genomic DNA with Mbo I or Sau3A, into the unique BamHI site. Digestion of recombinant cosmids with restriction enzymes that cleave frequently but do not disrupt the transcriptional promoters generates two small DNA templates for the synthesis of end-specific RNA probes to facilitate directional "walking." Cosmid restriction maps can be determined rapidly by one of several methods. The cosmids and methods we describe should have wide utility in determining the functional and structural organization of complex eukaryotic genomes and for physically linking distant genetic loci.
TL;DR: The use of RNA rather than DNA probes to map gene transcripts is presented in this volume, using probes as large as 2.5 kb to obtain transcription maps of new gene isolates that include 20-30 kb of genomic DNA.
Abstract: An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of the sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively
uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements. Methodology for nuclease mapping of RNA transcripts
with genomic DNA probes was first introduced by Berk and Sharp. We have adapted this technology for the purpose of obtaining transcription maps of new gene isolates that include 20-30 kb of genomic DNA, using probes as large as 2.5 kb. The information provided here will also be
useful for less demanding mapping experiments. The use of RNA rather than DNA probes to map gene transcripts is presented in this volume.
TL;DR: A number of RNA species of human papillomavirus types 6 and 11 from condylomata acuminata are identified and mapped by the electron microscopic R-loop technique and it is suggested that the papilloaviruses have sophisticated usage of ORFs through alternative promoters, mRNA splice sites, and polyadenylation sites.
Abstract: We have identified and mapped a number of RNA species of human papillomavirus types 6 and 11 from condylomata acuminata by the electron microscopic R-loop technique Each of the early (E)- and late (L)-region open reading frames (ORFs) deduced from the DNA sequences was represented in one or more transcripts In addition, RNA species that could encode the modulator of DNA replication and the repressor of transcription, functions recently identified in the genetically similar bovine papillomavirus type 1, were also detected Some ORFs were 5' proximal in one or more transcripts, whereas others were not 5' proximal in any species, suggesting that internal initiation of translation might be required to gain access to these latter ORFs Virtually all transcripts had their 5' ends located in the E region and were polyadenylated at one of two sites, ie, at the end of the E region or at the end of the L region The great majority of the RNAs were derived from the E region of the genome, with one species approximately 50 to 100 times more abundant than the others For most of the RNAs, the 5' end mapped near nucleotide 700; minor populations had 5' ends near nucleotide 100 or 1200 By correlating our mapping data with the genomic DNA sequences as well as available RNA structures and cDNA sequences of several papillomaviruses, we predict a number of mRNA splice donor and acceptor sites and suggest that the papillomaviruses have sophisticated usage of ORFs through alternative promoters, mRNA splice sites, and polyadenylation sites
TL;DR: Pulsed-field gel electrophoresis and "cosmid walking" have been used to establish a molecular map of the human major histocompatibility complex (MHC) and defined the positions of the genes for the tumor necrosis factors alpha and beta in the human MHC.
Abstract: Pulsed-field gel electrophoresis and "cosmid walking" have been used to establish a molecular map of the human major histocompatibility complex (MHC). We have isolated approximately equal to 230 kilobases (kb) of genomic DNA in overlapping cosmid clones covering the genes for the second and fourth components of complement (C2 and C4, respectively), factor B, and steroid 21-hydroxylase, and approximately equal to 82 kb of genomic DNA surrounding the genes for the tumor necrosis factors alpha and beta. Single-copy hybridization probes isolated from these cosmid clusters and probes for the known MHC gene loci were hybridized to Southern blots of genomic DNA that had been digested with infrequently cutting restriction endonucleases and separated on pulsed-field gels. The data obtained allowed the construction of a long-range genomic restriction map and indicated that the MHC spans 3800 kb. This map orients the MHC class III gene cluster with respect to the DR subregion; the C2 gene is on the telomeric side of the 21-hydroxylase B gene. In addition we have defined the positions of the genes for the tumor necrosis factors alpha and beta in the human MHC. Genes for the alpha chain of DR and 21-hydroxylase B are separated by at least 300 kb, while the distance between the genes for C2 and tumor necrosis factor alpha is 390 kb. The HLA-B locus lies approximately equal to 250 kb on the telomeric side of the tumor necrosis factor genes.
TL;DR: This work isolated 15 cDNA clones from the Okayama-Berg human cDNA library, and found two that can complement the tsBN2 mutation with an efficiency comparable to that of the genomic DNA clone.
Abstract: The human RCCI gene was cloned after DNA-mediated gene transfer into the tsBN2 cell line, which shows premature chromosome condensation at nonpermissive temperatures (39.5-40°C). This gene codes for a 2.5-kb poly(A) + RNA that is well conserved in hamsters and humans. We isolated 15 cDNA clones from the Okayama-Berg human cDNA library, and found two that can complement the tsBN2 mutation with an efficiency comparable to that of the genomic DNA clone. The base sequences of these two active cDNA clones differ at the 5' proximal end, yet both have a common open reading frame, encoding a protein of 421 amino acids with a calculated molecular weight of 44,847 and with seven homologous repeated domains of about 60 amino acids. This human RCC1 gene was located to human chromosome 1 using sorted chromosomal fractions.
TL;DR: Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestripes.
Abstract: A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.
TL;DR: The genomic DNA segment encoding alpha AGP is cloned and it is shown that it contains three adjacentalpha AGP coding regions, AGP‐A, B and B‘, identical in exon–intron organization but with slightly different coding potential, account for the heterogeneity observed by protein sequencing.
Abstract: alpha 1-acid glycoprotein (alpha AGP) is a well-characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA segment encoding alpha AGP and we show that it contains three adjacent alpha AGP coding regions, AGP-A, B and B', identical in exon--intron organization but with slightly different coding potential. These results account for the heterogeneity observed by protein sequencing. Southern blot analysis indicates that the cloned cluster contains all the alpha AGP coding sequences present in the human genome. The larger majority of alpha AGP mRNA in human liver is transcribed from AGP-A, whose promoter and cap site have been determined while the level of AGP-B and B' mRNA in human liver is very low. Using Hep3B hepatoma cells as a model system for the in vitro study of the acute phase reaction, we show that only AGP-A is strongly induced by treatment with culture medium of LPS stimulated monocytes.
TL;DR: Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase, demonstrating extensive homologies between the fetal Bt ChoEase encoded by these clones and other cholinesterases of various forms and species.
Abstract: To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ "true" acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from lambda gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.
TL;DR: Mouse genomic DNA contains multiple copies of sequences homologous to the Drosophila "Krüppel," a member of the "gap" class of developmental control genes of the fruit fly, which contains multiple finger-like folded domains capable of binding to nucleic acids.
TL;DR: Southern blot analysis of human genomic DNA indicated that TH is encoded by a single gene, which suggests that the four different forms of TH mRNA are produced by alternative RNA splicing from a single primary transcript.
TL;DR: The cDNA‐sequencing data show that this Shaker (IAchannel) gene can encode a protein of ˜35 kd with three α‐helical membrane‐spanning sequences near its carboxyl terminus with a striking homology with membrane‐ spanning sequences of the vertabrate Na+ channel.
Abstract: We have cloned 215-kb DNA containing the maternal effect region (ME) of the Shaker gene complex (shC) at 16F of the Drosophila X chromosome. Five translocation and deletion breakpoints have been mapped on the cloned DNA allowing a correlation of the genetic map to transcription units. The ME region spans ˜100 kb. The genetic behavior of this region correlates with the occurrence of maternal RNAs in this part of the ShC. Two transcripts have been identified in the vicinity of chromosomal rearrangements which cause a Sh phenotype. These are a 4.5-kb transcript interrupted by T(x;2)B27 and a 2-kb transcript interrupted by T(X;3)ShLC and T(X;Y)W32. The latter transcript is derived from a primary transcript which spans >65 kb genomic DNA. The cDNA-sequencing data show that this Shaker (IAchannel) gene can encode a protein of ˜35 kd with three α-helical membrane-spanning sequences near its carboxyl terminus. These have a striking homology with membrane-spanning sequences of the vertabrate Na+ channel.
TL;DR: The authors detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231.
Abstract: We have detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231. Hybridization of synthetic oligonucleotides specific for human Ki-ras sequences showed a mutation at codon 13. The polymerase chain reaction with Ki-ras specific amplimers revealed a guanosine to adenosine transition at the second position of codon 13, resulting in a substitution of glycine by aspartic acid. The codon 13 mutation is also detected in one Ki-ras allele of the MDA-MB231 cell line.
TL;DR: The isolation and nucleotide sequence of a full length CD7 cDNA, and of a cDNA for an unusual intron‐bearing precursor are described, which predicts a highly glycosylated membrane protein with homology to members of the immunoglobulin superfamily, and no relationship to known oncogenes.
Abstract: The human CD7 antigen (gp40) is a cell surface glycoprotein found on thymocytes and mature T-cells. It is one of the earliest antigens to appear on cells of the T-lymphocyte lineage, and the most reliable clinical marker of T-cell acute lymphocytic leukemia. This report describes the isolation and nucleotide sequence of a full length CD7 cDNA, and of a cDNA for an unusual intron-bearing precursor. The DNA sequence of the clone predicts a highly glycosylated membrane protein with homology to members of the immunoglobulin superfamily, and no relationship to known oncogenes. Over-expression of CD7 RNA was observed in only one T-cell tumor line, and genomic DNA rearrangement was not observed in any lines. Prompted by a recent suggestion that CD7 plays a role in IgM binding, COS cells expressing CD7 were tested and found not to bind IgM or IgM immune complexes.