[Chromosome sorting and the genomic DNA library].
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Chromosome Mapping↗
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Construction and characterization of a sheep BAC library of three genome equivalents.
A sheep BAC library of over three genome equivalents was constructed and arrayed in superpools and row, column, and plate pools. The library contains 90,000 clones distributed in 39 superpools. The average insert size was estimated at 123 kb. The library was screened by PCR with 77 primer pairs corresponding to ovine microsatellites distributed throughout the genome. The probability of finding a random sequence in the library could be estimated at 0.96.
Animals↗
Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.
A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.
Amino Acid Sequence↗
Molecular cloning and structural analysis of canine gastric H+,K(+)-ATPase.
Gastric hydrogen-potassium ATPase (H+,K(+)-ATPase) is a heterodimeric protein which participates in the formation of hydrochloric acid. We cloned canine H+,K(+)-ATPase alpha and beta subunit cDNAs from canine gastric cDNA libraries and the alpha subunit gene from a canine genomic library. The alpha subunit gene is 13 kb in length and contains 21 introns ranging from 77 to 1,076 bp. Its 5'-flanking region contains putative regulatory motifs for transcription that are similar to those found in H+,K(+)-ATPase genes from other species. The open reading frames of alpha and beta subunit cDNAs are 3,500 and 870 bp in length and encode proteins of 1,034 and 290 amino acids, respectively. They are 80-90% homologous to corresponding cDNAs previously identified in porcine and rodent gastric tissues.
Amino Acid Sequence↗
Characterization of a novel nodulin gene in soybean that shares sequence similarity to the gene for nodulin-24.
A gene encoding for nodulin-16 (N-16) was isolated from a soybean genomic library. Nucleotide sequence analysis of the cDNA and the genomic clone of N-16 indicated that the coding region of this gene is 330 bp long and is interrupted by a single intron of 494 bp. The coding region of the N-16 gene shows a high degree of localized sequence similarity with the coding sequence of soybean nodulin-24 (N-24). Sequence similarity between the two genes is limited to the coding region of 90 bp in the first exon and the first 54 bp in the second exon of the N-16 gene which is repeated as the 2nd, 3rd, and 4th exons in the N-24 gene. The N-24 gene has been postulated to be a result of repeated duplication of an insertion element consisting of the 54 bp exon and the flanking intron sequences. In the absence of sequence similarity in the regions flanking the 54 bp sequence between the N-16 and N-24 genes, the N-16 gene does not appear to be the ancestral gene. Both N-16 and N-24 have a similar hydrophobic amino terminal end suggesting that N-16 like N-24 is targeted to the peribacteroid membrane. Southern analysis of soybean genomic DNA shows the presence of other related sequences to the N-16 gene, one of which is found to be closely linked to it. Analysis of the temporal accumulation of the N-16 transcripts during nodule development in effective and ineffective nodules suggests that N-16 and related genes might differ from leghemoglobin and some other late nodulin genes in their mechanism of regulation.
Amino Acid Sequence↗
Structure of the human gene for the neural phosphoprotein B-50 (GAP-43).
The genomic DNA encoding the exons for the human neural phosphoprotein B-50 (GAP-43) was isolated using rat-based cDNA probes and oligonucleotides. Exons 2 and 3 were isolated from a genomic library, exon 1 was amplified by PCR on total genomic DNA. The gene consists of 3 exons and 2 large introns. The first exon encodes the N-terminal 10 amino acids of B-50 involved in membrane association of the protein. Exon 2 encodes the main part of the protein with the sites for protein kinase C-mediated phosphorylation and calmodulin binding, and includes a 10 amino acid residue insert not found in rodents. Exon 3 encodes the last 29 amino acid residues. The reported sequence extends the known cDNA structure to both the 5' and 3' ends. The 358 bp region upstream of the translational initiation codon, containing the main transcription starts, is purine-rich and does not include TATA or GC boxes. At the 3' end potential polyadenylation signals were found 510 bp and 584 bp downstream of the stopcodon in exon 3. The 5' end of the mRNA is heterogeneous in length, with primer extension products corresponding to a 5' untranslated region of 159 and 343 bases. Northern hybridizations, however, indicate that the majority of B-50 mRNA has a shorter 5' untranslated region, as was reported for the rat (Schrama et al., Soc. Neurosci. Abstr., 18 (1992) 333.4). The structural organization of the human gene is similar to that described for the rat (Grabczyk et al., Eur. J. Neurosci. 2 (1990) 822-827), and both translated and untranslated regions show a high degree of sequence homology to the rat gene.
Amino Acid Sequence↗
Biotin-labeled DNA probe in a PCR-based assay increases detection sensitivity for the equine hemoparasite Babesia caballi.
A DNA probe from Babesia caballi (Bc1) was selected by antibody screening of a genomic library. The Bc1 probe hybridized specifically to B. caballi genomic DNA. A polymerase-chain-reaction-based assay for B. caballi DNA was developed from primers deduced from the probe nucleotide sequence. An amplified product of 1.6 kb was detected from as little as 500 fg B. caballi template DNA. Sensitivity increased 1000-fold when the biotin-labeled Bc1 probe was hybridized to the amplicons in a Southern blot.
Animals↗
Olfactory marker protein gene: its structure and olfactory neuron-specific expression in transgenic mice.
Olfactory marker protein (OMP) genomic clones were isolated from a Charon 4A phage lambda rat genomic library. A 16.5-kilobase (kb) fragment of the rat genome containing the gene was isolated and characterized. Sequence analysis of the gene showed the absence of introns and the lack of CAAT and TATA boxes in the 5' flanking region. The transcription initiation site was mapped, and two sites 55 and 58 base pairs upstream of the ATG were observed. The 5' flanking region is rich in G+C residues and contains a G+C-rich motif as well as direct and inverted repeats. Functional OMP regulatory sequences were demonstrated in transgenic mice. An 11-kb chimeric gene was constructed in which the coding region for OMP was replaced with that for Thy-1.1. In Thy-1.2 mice carrying this transgene, Thy-1.1 was expressed solely by olfactory receptor neurons and their axons and terminals in the olfactory bulb.
Animals↗
Genomic basis for antifreeze peptide heterogeneity and abundance in an Antarctic eel pout: gene structures and organization.
Type III antifreeze protein (AFP) in the Antarctic eel pout (Lycodichthys dearborni) occurs as a heterogeneous family of three major and at least five minor variants collectively maintained at high year-round blood levels (> 20 mg/ml). Two major AFPs (RD1, RD2) are 7 kD in size, and the third (RD3) is 14 kD and is composed of two 7-kD AFP domains linked by a 9-residue connector. The genomic basis for the heterogeneity and abundance of these AFPs was investigated in this study. Genomic library screening statistics and restriction mapping analyses of 16 genomic clones together indicate an AFP gene family of over 40 genes, which would provide a sizable gene dosage for high AFP output. Two genomic clones, each containing 2 AFP genes, were characterized in detail. Three of the genes, were characterized in detail. Three of the genes encode the 7-kD AFP RD2, and are arrayed in direct 8.3-kb tandem repeats. The three gene sequences are nearly 100% identical for a distance of over 3 kb (inclusive of 1.7-kb 5' and 1-kb 3' flanking sequences), indicating strong selective pressure from the freezing Antarctic waters on maintaining functionality of AFP genes for producing adequate levels of AFPs for survival. The fourth AFP gene has multiple exons and translates into a multimeric AFP composed of at least six 7-kD AFP domains successively linked a 9-residue connector sequence similar to that of the dimeric 14-kD AFP, RD3. The presence of an unusually large (2.7-kb) AFP messenger RNA beside two small ones (0.9 kb and 0.7 kb) in Northern blot of liver RNA is consistent with the presence of a large functional multiexon AFP gene. Substantial sequence identity (83%) between the 1.2-kb introns of the multiexon AFP gene and the intron and 5' flanking sequences of RD2 genes suggests that the former could arise from recombinant events that linked two adjacent 7-kD AFP genes in the 8.3-kb tandem repeats followed by duplication events to produce the multiple exons.
Amino Acid Sequence↗
Genomic organization of the 5' region of the human thyroglobulin gene.
OBJECTIVE: The purpose of the present work is to establish the intron-exon organization from exon 12 to exon 23 of the human thyroglobulin gene and to construct a physical map of the 5' terminal half of the gene. DESIGN: Screening of a genomic library and subsequent restriction map, hybridization and sequencing methods have been employed to characterize the recombinant positive phages. METHODS: A human genomic DNA library was screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. Intron/exon junction sequences were determined by the Taq polymerase-based chain terminator method. Finally, the thyroglobulin gene was mapped using the Gene Bridge 4 radiation hybrid clone panel. RESULTS: We isolated and characterized four lambda phage clones that include nucleotides 3002 to 4816 of the thyroglobulin mRNA, encompassing exons 12 to 23 of the gene. The exon sizes range between 78 and 219 nucleotides. We found that the GT-AG splicing sequences rule was perfectly respected in all the introns. A total of 7302 intronic bases was analyzed. Hormogenic tyrosine 5 and 1291 are encoded by exons 2 and 18. Also, seven alternative spliced variants are associated with the 5' region. Thyroglobulin gene maps to 5,5 centiRays from the AFMA053XF1 marker, in chromosome 8. CONCLUSIONS: The present study shows that the first 4857 bases of thyroglobulin mRNA are divided into 23 exons and the four phages isolated include 32.6 kb genomic DNA, covering 1815 nucleotides of exonic sequence distributed in 12 exons, from exon 12 to 23.
5' Untranslated Regions↗
Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers.
We describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotide. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)n microsatellites with this approach contained clones with inserts containing CA repeats. We have also used this protocol for enrichment of (CAG)n and (AGAT)n sequence repeats and for Not I jumping clones. We have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.
Base Sequence↗
Identification of herpes simplex virus RNAs that interact specifically with regulatory protein ICP27 in vivo.
Herpes simplex virus type 1 (HSV-1) protein ICP27 has an essential regulatory role during viral replication, in part by post-transcriptional control of gene expression, and has a counterpart in all herpes viruses sequenced so far. Although much is known about the functions of this signature herpesvirus protein, little is known about its RNA binding capabilities; ICP27 interacts with specificity for a subset of intronless HSV-1 RNAs and poly(G), through its RGG box. We performed an in vivo yeast three-hybrid screen of an HSV-1 genomic library, searching for ICP27 interacting RNAs. Comparable with a yeast genomic screen, 24 of 55 single inserts mapped to antisense strands of HSV-1 transcribed regions or non-transcribed regions. The 31 HSV-1 sense RNAs identified were 35 to 225 nucleotides in length and interacted with preferred specificity for ICP27 as compared with an unrelated RNA-binding protein. They map to 10 monocistronic and 10 polycistronic transcripts of all kinetic classes and represent 28 open reading frames encoding predominantly essential viral proteins with roles in viral DNA replication and virion maturation. Several studies show regulatory effects by ICP27 on the majority of these transcripts, consistent with its regulation of the early-late switch in the HSV-1 life cycle. Deletion of the ICP27 RGG box and the ICP27 M15 mutation, both lethal in virus, abolished or severely reduced the ICP27-RNA interactions, indicating their biological relevance. The study facilitates continued study of gene regulation by ICP27 by further defining its interactions with viral RNAs.
Binding Sites↗
Generation of the primary antibody repertoire in rabbits: expression of a diverse set of Igk-V genes may compensate for limited combinatorial diversity at the heavy chain locus.
In mouse and human, generation of combinatorial diversity through use of different heavy and light chain variable region genes in immunoglobulin rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal, as only a few Igh-V genes are rearranged and expressed. To investigate the contribution of combinatorial diversity toward generation of the rabbit V(kappa) repertoire, we constructed five genomic libraries from rabbit kidney DNA and 1 cDNA library from the bone marrow of a 1-day-old rabbit using a series of polymerase chain reaction-based strategies. Our analyses indicate that most of the sequences that we recovered from our libraries belong to a single family and some are extremely similar. The actual number of germline Igk-V genes is potentially greater than our conservative estimate of at least 39, 28 of which we found expressed as mRNA. The germline Igk-V genes display different lengths of the coding region 3' of Cys 88 ranging from 7 to 12 amino acids, resulting in CDR3 length heterogeneity among functional V(kappa)J(kappa) sequences ranging from 8 to 15 amino acids. Some of the V(kappa)J(kappa) junctions had N and P nucleotide additions. Thus, in contrast to limited combinatorial diversity of its heavy chain, the rabbit can draw upon a diverse set of germline Igk-V genes. The kappa light chain has the potential to be a major contributor toward generation of the antibody specificities of the rabbit pre-immune repertoire.
Amino Acid Sequence↗
Cloning of the human and mouse type X collagen genes and mapping of the mouse type X collagen gene to chromosome 10.
Type X collagen, a homotrimer of alpha 1 (X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously described the isolation of a small fragment of the human type X collagen gene (COL10A1) and its localization to the q21-q22 region of human chromosome 6 [Apte, S., Mattei, M.-G. & Olsen, B. R. (1991) FEBS Lett. 282, 393-396]. Using this fragment as a probe to screen genomic libraries, we report here the isolation of human and mouse genomic clones which contain the major part of the human and mouse type X collagen genes. In both species, the 14-kb genomic clones which were isolated contain a long open reading frame (greater than 2000 bp in length) which codes for the entire C-terminal non-collagenous (NC1) domain, the entire collagenous (COL) domain and part of the N-terminal non-collagenous (NC2) domain of the alpha 1(X) collagen chain. The human genomic clone contains the major part of the COL10A1 gene, in addition to the region we have previously cloned, and is highly similar to the corresponding portions of the mouse genomic clone (84.5% similarity at the nucleotide level, and 86.1% at the level of the conceptual translation product). The identification of the mouse genomic clone as the alpha 1(X) collagen gene (Col10a1) was confirmed by in situ hybridization of a fragment of the mouse genomic clone to sections from newborn mice. Hybridization was restricted to the hypertrophic chondrocytes of developing chondroepiphyses, being absent in small chondrocytes and in other tissues. Using interspecific backcross analysis, the locus for the mouse alpha 1 (X) collagen gene was assigned to chromosome 10. The cloning and chromosomal mapping of the human and mouse alpha 1 (X) collagen genes now permit the investigation of the possible role of type X collagen gene defects in the genesis of chondrodysplasias in both species and provide data essential for the generation of transgenic mice deficient in type X collagen.
Amino Acid Sequence↗
Isolation of low-copy-number sequences that neighbor satellite DNA in mammals.
To investigate the role of satellite DNA in eukaryotic genomes, we isolated from an African green monkey (Cercopithecus aethiops) genomic library cloned segments containing the previously described deca-satellite linked to low-copy-number genomic sequences. Three such clones were obtained. The low-copy-number sequences in the three clones do not cross-hybridize suggesting that they derive from different genomic loci. The structure of one of the clones, lambda MkA, is described in detail. Subcloned segments containing the low-copy-number sequences from lambda MkA anneal to monkey, human and mouse genomic DNA. The subcloned probes were used to select clones containing homologous sequences from a second, independent monkey library as well as from human and mouse genomic libraries. Several of the newly isolated monkey clones hybridized to probes containing the species-specific deca- and alpha-satellites, confirming the genomic association of the low-copy-number sequence in lambda MkA with satellite DNA. Moreover, several of the human and mouse clones hybridized to species-specific human and mouse satellite DNAs, respectively. These experiments indicate that the low-copy-number sequence in lambda MkA and its association with satellite DNA is conserved in primates and rodents.
Animals↗
[Construction of a pufferfish gridded cosmid library].
OBJECTIVE: Pufferfish is a newly-established model organism in human genome research, which has been demonstrated its unique advantages in comparative genomics. Construction of a genomic library of pufferfish is the prerequisite to any studies of its genome. METHODS: Genomic DNA from Fugu rubripes, a species distribute only in Japan and China seas, was used to construct a library in a novel cosmid vector, named sCOGH2, which contains an exon-trapping cassette. RESULTS: This library is composed of 57,600 clones gridded in 60,096-well cell culture plates, one clone each well. The average size of the inserts is around 35 kb. It provides 99.2% probability to find any unique DNA fragment of pufferfish in this library. The clones of this library can survive after more than 10 times freeze-thaw, also demonstrating high stability in propagation. The clones shone highly positive signals when hybridized with the pufferfish genomic DNA. CONCLUSIONS: The library accords with qualitative demands of a cosmid library.
Animals↗
The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.).
We report the results of a study on the effectiveness of Cot filtration (CF) in the characterization of the gene space of bread wheat (Triticum aestivum L.), a large genome species (1C = 16,700 Mb) of tremendous agronomic importance. Using published Cot data as a guide, 2 genomic libraries for hexaploid wheat were constructed from the single-stranded DNA collected at Cot values > 1188 and 1639 M x s. Compared with sequences from a whole genome shotgun library from Aegilops tauschii (the D genome donor of bread wheat), the CF libraries exhibited 13.7-fold enrichment in genes, 5.8-fold enrichment in unknown low-copy sequences, and a 3-fold reduction in repetitive DNA. CF is twice as efficient as methylation filtration at enriching wheat genes. This research suggests that, with improvements, CF will be a highly useful tool in sequencing the gene space of wheat.
Cloning, Molecular↗
A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis.
The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously.
Base Sequence↗