Irma L. Pirtle

Dictation of the shape of mesoscale semiconductor nanoparticle assemblies by plasmid DNA

We have developed a method of semiconductor nanostructure fabrication relying on the size and shape of a polynucleotide to dictate the overall structure of an assembly of individual nanoparticles. This is exemplified by our use of the 3455‐basepair circular plasmid DNA molecule pUCLeu4 which, when anchored to a suitably derivatized substrate, yields an array of semiconductor nanoparticles matching the shape of the biopolymer stabilizer. The viability of the methodology was confirmed using data from high resolution transmission electron microscopy, selected area electron diffraction, and linear optical absorption spectroscopy. This is a unique demonstration of the self‐assembly of mesoscale semiconductor nanostructures using biological macromolecules as templates.

Characterization of the transcription unit and two processed pseudogenes of chimpanzee triosephosphate isomerase (TPI)

Three members of the chimpanzee TPI (encoding triosephosphate isomerase) gene family, the transcription unit and two processed pseudogenes, have been characterized by genomic blotting and nucleotide sequence analysis. The bona fide TPI gene spans 3.5 kb with seven exons and six introns, and is the first hominoid TPI gene to be completely sequenced. The chimpanzee gene exhibits a very high degree of sequence identity with human and rhesus TPI genes. For example, the polypeptides of 248 amino acids (aa) encoded by the chimpanzee and human TPI genes are identical, but the codons for five of these aa differ in the third codon wobble position. No alternative splice sites could be identified in the intervening sequences of the gene and, thus, the molecular basis for the synthesis of the proliferation-specific TPI isozyme observed in hominoids remains elusive. An Alu member occurs upstream from one of the processed pseudogenes, and short sequences with significant identity to the primate LINE-1 element flank the region encompassing the Alu member and TPI pseudogene. A solitary endogenous retroviral long terminal repeat occurs within the structural region of the other processed pseudogene. The ages of the processed pseudogenes are estimated to be 2.6 and 10.4 million years, implying that one was inserted into the genome before and one after the divergence of the chimpanzee and human lineages.

The nucleotide sequence of tyrosine tRNAQ∗ΨA from bovine liver

The nucleotide sequence of tyrosine tRNAQ∗ΨA from bovine liver was determined to be pC-C-U-U-C-m2G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3U-A-G-A-G-C-m22G-m22G-A-G-G-A-C-U-Q∗-Ψ-A-m1G-A-Ψm-C -C-U-U-A-G-m7G-D-m5C-G-C-U-G-G-T-Ψ-C-G-m1A-U-U-C-C-G-G-C-U-C-G-A-A-G-G-A-C-C-AOH. This tyrosine tRNA is 76 nucleotides in length, and contains two hypermodified nucleosides-—3-(3-amino-3-carboxylpropyl uridine (acp3U) and β-d-galactosylqueuosine (Q∗). The molecule also has a pseudouridine in the middle position of the anticodon, and is the first tRNA sequenced which has an adjacent pair of N2,N2-dimethylguanosine (m22G) residues.

Localization of three DNA segments encompassing tRNA genes to human chromosomes 1, 5, and 16: Proposed mechanism and significance of tRNA gene dispersion

The chromosomal locations of three cloned human DNA fragments encompassing tRNA genes have been determined by Southern analysis of human-rodent somatic cell hybrid DNAs with subfragments from these cloned genes and flanking sequences used as hybridization probes. These three DNA segments have been assigned to human chromosomes 1, 5, and 16, and homologous sequences are probably located on chromosome 14 and a separate locus on chromosome 1. These studies, combined with previous results, indicate that tRNA genes and pseudogenes are dispersed on at least seven different human chromosomes and suggest that these sequences will probably be found on most, if not all, human chromosomes. Short (8-12 nucleotide) direct terminal repeats flank many of the dispersed tRNA genes. The presence of these flanking repeats, combined with the dispersion of tRNA genes throughout the human genome, suggests that many of these genes may have arisen by an RNA-mediated retroposition mechanism. The possible functional significance of this gene dispersion is considered.

IBM microcomputer programs that analyze DNA sequences for tRNA genes

A set of four computer programs that search DNA sequence data files for transfer RNA genes have been written in IBM (Microsoft) BASIC for the IBM personal computer. These programs locate and plot predicted secondary structures of tRNA genes in the cloverleaf conformation. The set of programs are applicable to eukaryotic tRNA genes, including those containing intervening sequences, and to prokaryotic and mitochondrial tRNA genes. In addition, two of the programs search up to 150 residues downstream of tRNA gene sequences for possible eukaryotic RNA polymerase III termination sites comprised of at least four consecutive T residues. Molecular biologists studying a variety of gene sequence and flanking regions can use these programs to search for the additional presence of tRNA genes. Furthermore, investigators studying tRNA gene structure-to-function relationships would not need to do extensive restriction mapping to locate tRNA gene sequences within their cloned DNA fragments.

A human DNA segment encompassing leucine and methionine tRNA pseudogenes localized on chromosome 6.

A human genomic clone, designated LHtlm8, that strongly hybridized to a mammalian leucine tRNA(IAG) probe, was found to encompass a pair of tRNA pseudogenes that are transcribed in a homologous cell extract. A leucine tRNA(AAG) pseudogene (TRLP1) is 2.1-kb upstream and of opposite polarity to a methionine elongator tRNA(CAU) pseudogene (TRMEP1). TRLP1 has three nucleotide variations (97% identity) from its cognate leucine tRNA(IAG), while TRMEP1 has a 78% identity with its cognate tRNA. Similar to a number of other eukaryotic tRNA pseudogenes, presumptive precursor tRNA transcripts are generated from the two pseudogenes in vitro, but possibly due to their aberrant and unstable secondary and tertiary structures, no detectable mature tRNA products are observed. The two tRNA pseudogenes are encompassed within a 9.6-kb EcoRI fragment that has been assigned to the chromosomal locus, 6pter-q13, by Southern blot hybridization of human-rodent somatic cell hybrid DNAs with probes derived from the cloned tRNA pseudogenes and flanking sequences. A 4.4-kb EcoRI fragment also harbored in clone LHtlm8 was mapped to human chromosome 11, suggesting that the two EcoRI fragments were inadvertantly ligated together during construction of the genomic library.

Structural analysis of a bovine arginine tRNACCG gene

A bovine genomic clone containing a 17.4-kb DNA fragment was isolated and found to contain a solitary arginine tRNA gene with an anticodon of CCG that has a 100% identity to its cognate tRNA. This arginine tRNA gene, symbolized as TRR4, has a characteristic internal split promoter and a typical termination site for RNA polymerase III. The tRNA gene was transcribed in vitro by RNA polymerase III using a HeLa cell-free extract to yield a mature-sized tRNA product. The gene was mapped to bovine chromosome 19 using a panel of bovine-rodent somatic cell hybrid DNAs.