General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

Rabbit EPO gene and cDNA: expression of rabbit EPO after intramuscular injection of pDNA

Erythropoietin (EPO) cDNA was cloned from kidney total RNA of a NZW rabbit. The cDNA comprises a 588-bp open reading frame encoding a 195 amino acid protein with distinguishable regions of high of homology to other mammalian EPOs. Intramuscular injection of mice with a rabbit EPO expression plasmid resulted in a significant hematocrit increase. A rabbit genomic DNA fragment was also cloned using the rabbit EPO cDNA. This 4312-bp genomic DNA fragment contains sequences homologous to the mouse EPO promoter and hypoxia-responsive enhancer. In addition, the genomic DNA also presents a high degree of conservation to other regions involved in hypoxia response. Sequence divergence in the 3' UTR may indicate differences in regulation of mRNA stability or response to low oxygen tension.     

Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

Rapid identification of DNA fragments containing promoters for RNA polymerase II

We describe a direct procedure for screening genomic recombinant DNA libraries or restriction fragments of cloned DNA regions for RNA polymerase II promoters. Cellular polyadenylated mRNA is chemically de-capped by beta-elimination reaction and enzymatically re-capped with [alpha-32P]GTP by vaccinia guanylyl transferase. Since this enzyme only accepts di- or triphosphorylated 5' termini as a substrate, the mRNAs are labeled exclusively at the first nucleotide, irrespective of whether the mRNA was intact or fragmented before in vitro capping. By using in vitro-capped mRNA as a hybridization probe, recombinant DNA molecules or restriction fragments that carry a cap site (and thus likely an RNA polymerase II promoter) can directly be identified. Here, we demonstrate the applicability of this procedure by the isolation and characterization of several genomic DNA clones containing RNA polymerase II promoter sequences, that are highly active in liver.     

TECHNICAL REPORT: Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques

Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cells     

Glutathione S-transferase Ya subunit is coded by a multigene family located on a single mouse chromosome.

A cloned DNA probe of Ya, the major glutathione S-transferase subunit in rat liver, was used to study the organization of Ya genes in the mouse genome. Southern blot analysis of mouse genomic DNA indicates that the Ya subunit is encoded by a multigene family. The chromosomal distribution of Ya genes was determined by analysis of DNA from a panel of mouse-Chinese hamster somatic cell hybrids. All detectable Ya genes were found to be located on chromosome 9. At least some of the Ya-specific DNA sequences are clustered since, by screening a mouse genomic library, two recombinant phages, each containing two different Ya DNA sequences in the same insert, have been isolated. The finding that Ya is encoded by a cluster of different genes raises the question of the specificity of the different Ya DNA sequences.     

Characterization of cloned sequences complementary to F9 cell double-stranded RNA and their expression during differentiation

In a cDNA library prepared from the RNA of cultured murine F9 teratocarcinoma cells, we identified sequences exhibiting strong hybridization to double-stranded RNA (dsRNA) of F9 cells but weak hybridization to mouse liver dsRNA. Northern-blot hybridization of RNA extracted from F9 cells with or without treatment with retinoic acid revealed differences in the expression of some of these sequences in undifferentiated and differentiated cells. As shown by Southern-blot hybridization experiments, these differences of expression were not related to a gross rearrangement of the corresponding genomic DNA sequences of the differentiated cells. When RNA from F9 cells was used, one of the cloned dsRNA-related sequences selected mRNA which was translated in vitro to a polypeptide with an Mr of 24,000; the level of this mRNA was reduced in F9 cells that had been treated with retinoic acid. Our results show that the differentiation of F9 cells induced by retinoic acid results in the differential expression of some middle-repetitive sequences.     

Gene transfer, expression, and molecular cloning of the human transferrin receptor gene

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk- cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5' end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3' untranslated sequences that are not required for receptor expression in L cells.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.     

Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.