Gene Transfer and Cloning of the Amino-Acid Transport System L from Human Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72389/1/j.1749-6632.1985.tb14893.x.pd

A multisite-directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene

A method to introduce multiple mutations and to reconstruct genes, using a single oligodeoxyribonucleotide and DNA polymerase with high processivity, such as modified T7 DNA polymerase [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 84 (1987a) 4767-4771], is described. A eukaryotic cDNA, coding for porcine growth hormone (pGH), was reconstructed in this study to delete 75 bp and to introduce a G --> A transition. The deletion removes 75 bp and brings an ATG just upstream from the codon for the first amino acid in the mature protein. Moreover, the G --> A substitution creates a new PvuII restriction site to facilitate further manipulation of the gene. Maximum mutation frequency with this multisite-directed mutagenesis is reached within 15min with an efficiency approaching 50%, when using the modified T7 DNA polymerase. No multisite-directed mutants were obtained when T4 DNA polymerase or Klenow (large) fragment of DNA polymerase I were used. The described method is also applicable to simple single site-directed mutations as well as to more complex gene reconstruction strategies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27137/1/0000130.pd

Eliminated sequences with different copy numbers clustered in the micronuclear genome of Tetrahymena thermophila

As the ciliated protozoan Tetrahymena thermophila develops a new macronucleus (MAC) from products of its micronucleus (MIC), several repetitive sequences are eliminated from the MAC genome. Four MIC DNA clones containing repetitive sequences that are eliminated from the MAC were obtained. One clone contains a representative from each of three families of eliminated sequences. One, present in 200–300 copies in the MIC, is almost completely eliminated from the MAC. A second, present in approximately 50 copies in the MIC, is scattered throughout the genome, although up to half of the family members examined could be localized to chromosome 2. Approximately one tenth of the members of this less repetitive family persist in the MAC while the rest are eliminated. The third type of eliminated sequence has three to four members, all of which are eliminated from the MAC. Three of the members are located on three of the five MIC chromosomes, and one could not be mapped. This sequence is clustered with the other two families of sequences in at least three of the four sites. All three types of eliminated sequences are found in similar arrangements in the MIC of several different inbred strains of T. thermophila .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47561/1/438_2004_Article_BF00397988.pd

Sequence and evolution of the regions between the rrn operons in the chloroplast genome of Euglena gracilis bacillaris

The rRNA genes are arranged in three sequential operons preceded by a fourth partial operon. Part or all of a 1462 nucleotide sequence extending from within the 3′-end of the 23S rRNA gene, across the 5S rRNA gene and a presumptive transcription terminator, to within the first structural gene (for 16S rRNA) of the rrn operon was determined for each region between operons. Homologies of the 3′-end of the 23S rRNA gene with the 4.5S rRNA genes of higher plant chloroplasts, and of the 5S rRNA gene with other 5S rRNA genes were examined. The region preceding the 16S rRNA gene, which is expected to contain sites for initiation and regulation of rrn transcription, includes a 305 base-pair sequence with substantial homology with structural genes elsewhere in the chloroplast genome. The homologies suggest that this portion of the leader evolved from copies of parts of the structural genes which had been inserted before the 16S rRNA genes. Thus the chloroplast rrn leader may provide a unique opportunity to study how a regulatory sequence evolved from well-defined structural genes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47554/1/438_2004_Article_BF00425555.pd

Location of a phenylalanine tRNA gene on the physical map of the Euglena gracilis chloroplast genome

A tRNAphe from Euglena gracilis bacillaris chloroplasts was purified and hybridized with restriction fragments of chloroplast DNA. From the hybridization pattern, the location of the corresponding gene on the physical map of the chloroplast genome was determined.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24051/1/0000301.pd

A Third Approach to Gene Prediction Suggests Thousands of Additional Human Transcribed Regions

The identification and characterization of the complete ensemble of genes is a main goal of deciphering the digital information stored in the human genome. Many algorithms for computational gene prediction have been described, ultimately derived from two basic concepts: (1) modeling gene structure and (2) recognizing sequence similarity. Successful hybrid methods combining these two concepts have also been developed. We present a third orthogonal approach to gene prediction, based on detecting the genomic signatures of transcription, accumulated over evolutionary time. We discuss four algorithms based on this third concept: Greens and CHOWDER, which quantify mutational strand biases caused by transcription-coupled DNA repair, and ROAST and PASTA, which are based on strand-specific selection against polyadenylation signals. We combined these algorithms into an integrated method called FEAST, which we used to predict the location and orientation of thousands of putative transcription units not overlapping known genes. Many of the newly predicted transcriptional units do not appear to code for proteins. The new algorithms are particularly apt at detecting genes with long introns and lacking sequence conservation. They therefore complement existing gene prediction methods and will help identify functional transcripts within many apparent “genomic deserts.

Organization of the chloroplast ribosomal RNA genes of Euglena gracilis bacillaris

The order of eight of the 29 endonuclease EcoRI-generated fragments of chloroplast DNA was determined. Three sets of rRNA genes aligned sequentially in the same orientation form part of this region. The repeated sets differ in the length and sequence of the spacers among themselves and with the rRNA genes of E. gracilis strain Z .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47546/1/438_2004_Article_BF00433298.pd

Intragenic complementation between Escherichia coli trp repressors with different defects in the tryptophan-binding pocket

Site-directed mutagenesis of the trpR gene (encoding the trp repressor, TrpR) was used to replace Gly85 with tryptophan (Trp or W), in order to place Trp near its normal location in the -tryptophan(-W)-binding pocket. The resulting mutant protein (G85W) exhibits weak, but significant repressor activity in vivo that is independent of the presence of -W in the media. This mutant negatively complements the chromosomal wild type (wt), but does not negatively complement either the wt or the super-repressor, E49K, when any of these alleles is expressed on a multicopy plasmid. Activity of the mutant repressor, G85W, when produced in vivo together with T44M, approaches that of the wt repressor. This result presumably reflects complementation between the two mutant polypeptides. Similar results are obtained when G85R or G85K are combined with T44M in vivo, but not when G85W is replaced by G85E. The level of repression is dependent on the presence of -W in the media. The TrpR with two mutations altering both Gly85 (G85W, G85R, G85E or G85K) and Thr44 (T44M) has no repressor activity. These results suggest a type of site-specific intragenic complementation where only certain alterations at Gly85 complement T44M. In this study, a positive charge or an indole ring appears to be required for the observed intragenic complementation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29909/1/0000266.pd

Organization of the chloroplast ribosomal RNA genes of Euglena gracilis bacillaris

The order of eight of the 29 endonuclease EcoRI-generated fragments of chloroplast DNA was determined. Three sets of rRNA genes aligned sequentially in the same orientation form part of this region. The repeated sets differ in the length and sequence of the spacers among themselves and with the rRNA genes of E. gracilis strain Z. © 1979 Springer-Verlag