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H G Morrison

Publications and source records attributed to H G Morrison.

At least 19 recordsLinked to original sources
PubMed2001

Three retrotransposon families in the genome of Giardia lamblia: two telomeric, one dead.

Transposable elements inhabiting eukaryotic genomes are generally regarded either as selfish DNA, which is selectively neutral to the host organism, or as parasitic DNA, deleterious to the host. Thus far, the only agreed-upon example of beneficial eukaryotic transposons is provided by Drosophila telomere-associated retrotransposons, which transpose directly to the chromosome ends and thereby protect them from degradation. This article reports the transposon content of the genome of the protozoan Giardia lamblia, one of the earliest-branching eukaryotes. A total of three non-long terminal repeat retrotransposon families have been identified, two of which are located at the ends of chromosomes, and the third one contains exclusively dead copies with multiple internal deletions, nucleotide substitutions, and frame shifts. No other reverse transcriptase- or transposase-related sequences were found. Thus, the entire genome of this protozoan, which is not known to reproduce sexually, contains only retrotransposons that are either confined to telomeric regions and possibly beneficial, or inactivated and completely nonfunctional.

Animals↗
PubMed2001

Giardia lamblia expresses a proteobacterial-like DnaK homolog.

We identified a novel gene encoding molecular chaperone HSP70 in the amitochondriate parasite Giardia lamblia. The predicted protein is similar to bacterial DnaK and mitochondrial HSP70s. The gene is transcribed and translated at a constant level during trophozoite growth and encystation. Alignment of the sequence with a data set of cytosolic, endoplasmic reticulum (ER), mitochondrial, and DnaK HSP70 homologs indicated that the sequence was extremely divergent and contained insertions unique to giardial HSP70s. Phylogenetic analyses demonstrated that this sequence was distinct from the cytosolic and ER forms and was most similar to proteobacterial and mitochondrial DnaKs. However, a specific relationship with the alpha proteobacterial and mitochondrial sequences was not strongly supported by phylogenetic analyses of this data set, in contrast to similar analyses of cpn60. These data neither confirm nor reject the possibility that this gene is a relic of secondary mitochondrial loss; they leave open the possibility that it was acquired in a separate endosymbiotic event.

Amino Acid Sequence↗
PubMed2000

The Giardia genome project database.

The Giardia genome project database provides an online resource for Giardia lamblia (WB strain, clone C6) genome sequence information. The database includes edited single-pass reads, the results of BLASTX searches, and details of progress towards sequencing the entire 12 million-bp Giardia genome. Pre-sorted BLASTX results can be retrieved based on keyword searches and BLAST searches of the high throughput Giardia data can be initiated from the web site or through NCBI. Descriptions of the genomic DNA libraries, project protocols and summary statistics are also available. Although the Giardia genome project is ongoing, new sequences are made available on a bi-monthly basis to ensure that researchers have access to information that may assist them in the search for genes and their biological function. The current URL of the Giardia genome project database is www.mbl.edu/Giardia.

Animals↗
PubMed1999

Cloning and sequencing of an acetyl-CoA synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.

A Giardia lamblia gene, Glacs, was cloned, sequenced and expressed in Escheria Coli. This gene codes for a 726 residue long acetyl-CoA synthetase (ADP-forming). This enzyme is responsible for the formation of acetate, a metabolic endproduct of G. lamblia. It is known from only two Type I amitochondriate eukaryotes, G. lamblia and Entamoeba histolytica and from the archaebacterium, Pyrococcus furiosus. With Glacs as query, homologous unidentified open reading frames were detected in the complete genomes of only a few archaebacteria and eubacteria. These form a new protein family present in all three domains of life, which probably plays a central role in the acyl-CoA metabolism but is of restricted taxonomic distribution.

Animals↗
PubMed1999

Primary structure and phylogenetic relationships of a malate dehydrogenase gene from Giardia lamblia.

The lactate and malate dehydrogenases comprise a complex protein superfamily with multiple enzyme homologues found in eubacteria, archaebacteria, and eukaryotes. In this study we describe the sequence and phylogenetic relationships of a malate dehydrogenase (MDH) gene from the amitochondriate diplomonad protist, Giardia lamblia. Parsimony, distance, and maximum-likelihood analyses of the MDH protein family solidly position G. lamblia MDH within a eukaryote cytosolic MDH clade, to the exclusion of chloroplast, mitochondrial, and peroxisomal homologues. Furthermore, G. lamblia MDH is specifically related to a homologue from Trichomonas vaginalis. This MDH topology, together with published phylogenetic analyses of beta-tubulin, chaperonin 60, valyl-tRNA synthetase, and EF-1alpha, suggests a sister-group relationship between diplomonads and parabasalids. Since these amitochondriate lineages contain genes encoding proteins which are characteristic of mitochondria and alpha-proteobacteria, their shared ancestry suggests that mitochondrial properties were lost in the common ancestor of both groups.

Amino Acid Sequence↗
PubMed1998

Sequence and phylogenetic position of a class II aldolase gene in the amitochondriate protist, Giardia lamblia.

A Giardia lamblia gene, Glfba, was cloned and sequenced. This gene codes for a 324-residue-long putative class II fructose-1, 6-bisphosphate aldolase. The positions of gaps and phylogenetic analysis with maximum likelihood and maximum parsimony methods showed the sequence to be most closely related to the as-yet uncharacterized aldolases of Helicobacter pylori and Aquifex aeolicus and to the group that comprises the Calvin-cycle aldolases of photosynthetic proteobacteria and cyanobacteria. In combination with the known taxonomic and functional distribution of class I and II aldolases, the results indicate that the G. lamblia enzyme is distinct in its evolutionary history from all eukaryotic fructose-1, 6-bisphosphate aldolases studied so far.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of CYP1A from the estuarine fish Fundulus heteroclitus and phylogenetic analysis of CYP1 genes: update with new sequences.

Since we published a phylogenetic analysis of the CYP1A subfamily in 1995, several additional full-length sequences have been reported, including three members of an entirely new subfamily, CYP1B. Two avian sequences were recently published, so that CYP1A sequence data are now available from three of the five major vertebrate lineages. The two new branches that have been added to the CYP1 family tree significantly add to our understanding of P450 evolution. The inclusion of the CYP1Bs to the phylogenetic analysis allows us to root inferred trees. Addition of the avian CYP1As indicates that the CYP1A1/CYP1A2 duplication present in the mammalian lineage may have occurred after the divergence of birds and mammals. The number of fish species from which full-length coding regions of CYP1A genes have been sequenced has increased from four (trout, plaice, toadfish, and scup) to nine. These include CYP1A sequences from tomcod, butterflyfish, sea bream, sea bass, and the full-length sequence of CYP1A from the killifish Fundulus heteroclitus that is reported here. Phylogenetic analyses incorporating the new fish CYP1A sequences support our original conclusion that the fish CYP1As are monophyletic and indicate that the genes are evolving at very different rates in different species.

Amino Acid Sequence↗
PubMed1996

Ancestral relationships of the major eukaryotic lineages.

Molecular systematics has revolutionized our understanding of microbial evolution. Phylogenetic frameworks relating all organisms in this biosphere can be inferred from comparisons of slowly evolving molecules such as the small and large subunit ribosomal RNAs. Unlike today's text book standard, the "Five Kingdoms" (plants, animals, fungi, protists and bacteria), molecular studies define three primary lines of descent (Eukaryotes, Eubacteria, and Archaebacteria). Within the Eukaryotes, the "higher" kingdoms (Fungi, Plantae, and Animalia) are joined by at least two novel complex evolutionary assemblages, the "Alveolates" (ciliates, dinoflagellates and apicomplexans) and the "Stramenopiles" (diatoms, oomycetes, labyrinthulids, brown algae and chrysophytes). The separation of these eukaryotic groups (described as the eukaryotic "crown") occurred approximately 10(9) years ago and was preceded by a succession of earlier diverging protist lineages, some as ancient as the separation of the prokaryotic domains. The molecular phylogenies suggest that multiple endosymbiotic events introduced plastids into discrete eukaryotic lineages.

Animal Population Groups↗
PubMed1995

Identification of V3 mutations that can compensate for inactivating mutations in C4 of simian immunodeficiency virus.

A valine to isoleucine substitution at position 322 within variable region 3 (V3) of envelope of simian immunodeficiency virus was previously shown to compensate for an inactivating valine to glycine mutation at position 448 in constant region 4 (C4) (Morrison et al., Virology 195, 167-174, 1993). Cloned DNA fragments with inactivating C4 mutations were combined with complex mixtures of mutant V3 sequences, and full length genomes were transfected into COS-1 cells. By cocultivating transfected cells with CEM x 174 cells, we were able to identify two additional compensatory V3-C4 combinations. Changing 334 proline to leucine compensated for an inactivating 428 asparagine to lysine mutation and changing 324 isoleucine to leucine compensated for an inactivating 448 valine to glycine mutation. The double mutants replicated efficiently in CEM x 174 cells, rhesus monkey peripheral blood mononuclear cells, and the continuously growing rhesus monkey T cell line 221. Surprisingly, the 324 I-->L and 33 P-->L mutations by themselves impaired SIVmac239 wild-type replication in CEM x 174 cells. These results confirm the cooperation between V3 and C4 sequences and they define additional specific residues participating in this cooperation.

Amino Acid Sequence↗
PubMed1995

Effects of mutations in constant regions 3 and 4 of envelope of simian immunodeficiency virus.

Twenty-six mutant forms of simian immunodeficiency virus strain mac239 were constructed with changes in constant region 4 (C4) of env. Twenty-four of these had a single amino acid change, one had changes in two amino acids, and one had a deletion of eight amino acids. The effects of these mutations on viral replication, gp160 processing, and binding of env protein to soluble CD4 receptor were analyzed. The C4 region was relatively sensitive to sequence changes since only 11 of the 26 mutants replicated appreciably. Eight of the 15 mutants that were replication incompetent exhibited grossly defective processing of the gp160 env precursor; these mutations likely resulted in global effects on gp160 structure. Six of the replication incompetent mutants exhibited normal or near normal gp160 processing and binding of env protein to sCD4 and thus were probably blocked at some step subsequent to binding of virus to its CD4 receptor. Only one of the C4 mutations, 441W-->R, resulted in greatly decreased binding to sCD4 while retaining normal processing of gp160. The equivalent residue in HIV-1 has similarly been shown previously to be important for binding of HIV-1 to the CD4 receptor. Since a W-->S mutation at position 441 in C4 of SIVmac239 affected both gp160 processing and sCD4 binding, it is not clear whether the 441 tryptophan is actually important for contacting CD4 or for maintaining an appropriate configuration. mutations within a highly conserved GGDPE sequence in C3 of SIVmac239 specifically affected CD4 binding, which is also similar to previous findings with HIV-1. These results demonstrate similar sequence requirements in SIVmac and HIV-1 env for binding C4, but they raise doubts as to whether C4 sequences are directly involved in the binding.

Amino Acid Sequence↗
PubMed1995

Identification of cytochrome P-450 1A (CYP1A) genes from two teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops), and phylogenetic analysis of CYP1A genes.

Cytochrome P-450-mediated responses to environmental challenges are well known in diverse animal taxa, but the evolution of the complex gene superfamily coding for these enzymes is poorly understood. Here we report a phylogenetic analysis of the cytochrome P-450 1A (CYP1A) genes including two new sequences determined from teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops). Degenerate PCR primers were used to amplify a 1.2 kbp fragment from liver cDNA. The toadfish PCR product was used as a probe to identify a full-length CYP1A clone from a toadfish liver cDNA library. The entire coding region of the scup CYP1A was obtained by rapid amplification of cDNA ends (RACE) using specific primers based on the sequence of the partial PCR product. The predicted protein sequences for toadfish and scup CYP1A shared 78% and 83% amino acid identity with rainbow trout CYP1A1 respectively. Amino acid identity with mammalian CYP1A proteins ranged from 51 to 60% for 505 aligned positions. Phylogenetic analysis of four teleost fish CYP1A genes (trout, toadfish, scup and plaice) and 12 mammalian CYP1A genes suggests a monophyletic origin of the teleost genes, with the trout gene being most divergent, and indicates three distinct groupings: mammalian 1A1, mammalian 1A2, and fish 1A. This supports the idea that the gene duplication event which gave rise to CYP1A1 and CYP1A2 occurred after the divergence of the lines leading to mammals and fish. These results establish a molecular phylogeny within the CYP1A subfamily, the first such detailed phylogenetic analysis within a cytochrome P-450 family.

Amino Acid Sequence↗
PubMed1994

SIVmac expressing hybrid envelope proteins containing HIV-1 V3 and/or C4 sequences is not competent for replication.

The V3- and C4-coding regions in the envelope gene of the infectious, pathogenic SIVmac239 clone were replaced by the corresponding HIV-1 sequences. Viral particles were obtained after transfection of COS-1 cells. Chimeric SIVmac constructs were not replication competent in the human T cell lines CEMx174, AA2, H9, and MT-4 or in primary cultures of rhesus monkey peripheral blood mononuclear cells. The lack of infectivity of the hybrid constructs was associated with inefficient proteolytic processing of the gp160env precursor. Unlike the modular nature of some proteins, gp120 appears to be a highly ordered molecule whose function is dependent on the integration of many discontinuous, interactive regions.

Amino Acid Sequence↗
PubMed1993

Evidence for the cooperation of gp120 amino acids 322 and 448 in SIVmac entry.

The substitution of glycine for valine at amino acid 448 in the C4 domain of SIVmac239 envelope glycoprotein gp120 resulted in little or no viral infectivity for CEMX174 cells and rhesus monkey peripheral blood mononuclear cells. The block to viral replication was located at an early stage including virus entry into cells since the mutant virus was severely impaired in its ability to form newly synthesized viral DNA during the initial 14 hr after exposure to cells. Envelope glycoprotein with the 448V-->G mutation bound to soluble CD4 in a manner similar to wild type in an in vitro assay and was also translated, processed, and cleaved in a manner similar to wild-type envelope protein. When CEMx174 cells were transfected with 448V-->G mutant proviral DNA and held for longer than 40 days, a variant appeared in one culture that was able to replicate with near wild-type kinetics. Sequencing of viral DNA from cultures infected with this variant revealed that the original 448V-->G mutation was retained in eight of eight clones analyzed but that six of eight clones analyzed had an additional mutation of V-->I at amino acid 322 of gp120. Amino acid 322 in SIVmac gp120 is located in a region which corresponds to V3 of HIV. Analysis of site-specific mutants demonstrated that the mutation of V-->I at position 322 can indeed compensate for the 448V-->G mutation, resulting in efficient virus entry and replication by the double mutant. These results identify single amino acids in gp120 that are critical for early events in SIVmac replication and suggest that the "V3" and C4 domains of SIVmac gp120 cooperate to effect virus entry.

Amino Acid Sequence↗
PubMed1993

A PCR-based strategy for extensive mutagenesis of a target DNA sequence.

A mixed population of mutagenic oligonucleotide primers was used to generate a set of point mutations in a short region of a retroviral gene by PCR amplification. The mixed population of mutagenic primers was generated by incorporating a mixture of A, G, C and T at specific sites during oligonucleotide synthesis. With the proportions of mutagenic nucleotides used for our experiments, 47 percent of the 213 clones analyzed had one or more point mutation in the target DNA sequence. In addition, unpredicted mutations were observed that contributed to the mutagenic complexity of the population. We have found this approach to be an efficient means for extensive mutagenesis of a defined target DNA sequence.

Amino Acid Sequence↗
PubMed1991

Simultaneous expression of the Lassa virus N and GPC genes from a single recombinant vaccinia virus.

A new transfer vector was constructed that directs the insertion of two heterologous genes into the vaccinia virus thymidine kinase (TK) gene during a single recombination event. This vector, pDAVAC2, contains bidirectional vaccinia P7.5 early/late promoter elements and two unique cloning sites. cDNA clones containing the complete coding sequences for the Lassa virus (Josiah strain) nucleoprotein (N) and glycoprotein (GPC) genes were inserted into the vaccinia TK gene using this transfer vector. The recombinant virus, V-LSGN-II, expressed proteins in cell culture that appeared to be authentic with respect to electrophoretic mobility, glycosylation, and post-translational cleavage. Indirect immunofluorescence (IFA) of recombinant virus-infected cells demonstrated both the bright granular and diffuse patterns of staining characteristic of the Lassa nucleoprotein and glycoprotein, respectively. Electron-dense inclusion bodies typical of arenavirus-infected cells were observed by electron microscopy in V-LSN and V-LSGN-II-infected cells, but not in V-LSGPC-infected cells. Mice inoculated with V-LSGN-II by intraperitoneal injection developed serum antibodies that reacted with authentic Lassa proteins in immunofluorescence and radioimmune precipitation assays. This recombinant virus represents an additional candidate for a Lassa fever vaccine and demonstrates the feasibility of expressing any two genes of interest in a single recombinant vaccinia virus through the use of the transfer vector pDAVAC2.

Animals↗