Meetings and Papers

     One of the pleasures of being a research scientist is going to meetings.  There you have the opportunity to meet others in your feild, discuss works in progress, hear what's new and what's hot, and have a little fun in the process.  Some of the meetings are in famous research centers like Cold Spring Harbor in New York.  Others are in the Rocky mountains or maybe even Europe.  Here is a selection of meetings that I have attended and presented my research at in the last decade or so, along with the abstracts of the presentations.
    Most of these presentations were works in progress of research that has since been published.  References for my published papers and their abstracts are here.

Meeting:  Gordon Research Conference on Mitochondria and Chloroplasts,
August 2002, Oxford, England

Growth Rate Dependent Structures of Wild-type and Mutant Mitochondrial DNA in the Yeast
Bojana Stevic, Julia Sable, Heather E. Lorimer
 There are four proposed mechanisms of mitochondrial DNA (mtDNA) replication in the yeast  Saccharomyces cerevisiae, each of which should produce distinctive structures in replicating DNA.  These are: 1) standard bi-directional replication from a replication origin, which should produce double-stranded bubbles and Y-shaped structures, 2) the D-loop model, which should produce a single stranded "loop" over the region of initial DNA replication, 3) recombination-dependent replication, which should produce complex cross-linked structures with the possibility of some single stranded regions arising from strand invasion and branch migration, and 4) rolling-circle replication, which should produce circles with long tails attached, potentially double-stranded, single-stranded or a combination depending on the mechanism of initiation on the second strand.  Previous work has provided evidence for the first three of these mechanisms in yeast.  We have used Brewer-Fangman 2-D gel electrophoresis to analyze mtDNA structures in wild-type (rho+) and deletion mutant (rho-) strains.  We have observed a continuous distribution of double stranded linear DNA in all cases.  Single-stranded DNA was found in all log phase cells, but only in slight amounts, if any, in stationary cells.  Rho- strains also exhibit discrete circular forms, both relaxed and supercoiled.  The rho+ genome produced some evidence of simple circular forms under our conditions, and we have observed an intriguing complex structure that is at least partially single-stranded.  Together with the finding of long single-stranded DNA molecules this may provide evidence for rolling circle DNA replication in yeast mtDNA.

Meeting:  Eukaryotic DNA Replication
Date:  Sept. 15 - 19, 1999

Location:  Cold Spring Harbor Laboratory,               Heather and Julia at CSH Sept. 1999
Cold Spring Harbor, NY.

Structural Analysis of Replicating Forms of Mitochondrial DNA in Yeast.
Julia E. Sable and Heather E. Lorimer. Youngstown State University, Youngstown OH, 44555

    Mitochondrial DNA (mtDNA) in the yeast Saccharomyces cerevisiae has a circular genetic map but has been shown to exist as both heterogenous linear and circular forms in vivo. We have used the Fangman/Brewer 2-D gel electrophoresis system to analyze mtDNA from cells containing both wild-type (rho+) and deletion mutant (rho-) mitochondrial genomes. A surprisingly complex array of forms of mtDNA was observed in rho- cells, including linear double stranded molecules in a continuous size range from a few hundred base pairs up to at least 20,000 kb. Circular molecules of varying superhelicity were also observed. The circular forms range from 1N through very large molecules, but only in genome sized increments, not continuous sizes as was observed in the linear molecules. In addition, a continuous range of linear ssDNA molecules were observed in rho+ as well as rho- cells. Unlike the other forms ssDNA is dependent on the cells growth rate with stationary cultures of both rho+ and rho- exhibiting little or no ssDNA. In rho- cells mtDNA can be maintained in the absence of the mitochondrial RNA polymerase, RPO41, but no SSDNA has been observed under these conditions. These results imply that there may be two separate mechanisms of mtDNA replication in yeast, one requiring RNA polymerase and potentially involving rolling circle replication.

Meeting: First International Feline Genetic Disease Conference
Date: June 25-29, 1998
Location: University of Pennsylvania, Philidelphia, PA

Talk: Coat Genetics in Domestic Cats
Heather E. Lorimer, Dept. of Biological Sciences
Youngstown State University, Youngstown, OH 44555

    Artificial selection by humans has resulted in preservation and proliferation of a large variety of coat colors, length, and texture, in the domestic cat.  Currently approximately fifteen genes, each with two or more alleles, cause easily observable differences.  In additional, there are polygenetic factors that affect coat color.
Color and coat genes encompass a wide variety of modes of inheritance.  These include the familiar sex-linked orange/red color which produces textbook mosaicism in heterozygous females, as well as simple dominant/recessive alleles such as the dilute gene which affects pigment deposition in the cells of the hair shaft, easily visible under low power magnification of individual hairs.  Other color genes are pleiotropic, causing a number of effects besides color.  The temperature sensitive allele of the albino gene causing pointed color in many cats including Siamese, effects the optic nerve pathways.  Dominant white is pleiotropic, epistatic, and has variable expression.  White color apparently results from altered development, particularly programmed cell death, and occasionally causes deafness through hair cell degeneration in the inner ear after birth.  The rex genes cause similar phenotypes of curly coats through different genes.  Tabby patterns were thought to be created by several alleles of one gene. Data collected from the cat fancy indicates that tabby patterns are probably caused by at least three separate genes with multiple alleles.
When the genes that are involved in coat color are located on the feline chromosomes they will provide genetic markers whose linkage to other genes should be easy to observe and thus they should contribute markedly to the feline genome project..

Poster: Hereditary Lymphosarcoma in Oriental Shorthair Cats
Heather E. Lorimer, Dept. of Biological Sciences
Youngstown State University, Youngstown, OH 44555

    Incidence of mediastinal lymphosarcoma in young FeLV-negative pedigreed Oriental Shorthair cats has been observed since the early 1980's.  Affected cats are typically between 3 months and 2 years of age, and present initially with dyspnea or difficulty in eating.  Characteristically further examination reveals a large mediastinal mass with metastases to surrounding lymph nodes.  Tumors grow rapidly and are quickly terminal unless treated.  Tumors regress rapidly in response to chemotherapy  and there are currently a number of affected cats that have survived for a year or more with treatment.
Pedigree analysis reveals that the vast majority of affected Oriental Shorthair's are descendants, on both their maternal and paternal sides, of a single male who died of lymphosarcoma in the early 1980's.  A few affected cats have this line of descent form only one parent.  In spite of those individuals who are not obviously inbred on the propositus, this lymphosarcoma has many attributes of a recessive genetic disease:  most affect cats trace back to the propositus on both sides, many generations of unaffected cats can be the ancestors of an affected litter, usually only approximately 1/4 of the kittens in a litter are affected, and when a cat that later develops the disease is bred to a relative, much larger percentages of kittens in the litter are affected.

Meeting: Eukaryotic DNA Replication
Date:  Sept. 3 -Sept. 7, 1997
Location: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Mechanisms of Human Mitochondrial DNA Replication.
Holt, I. J.1, Lorimer, H. E.2, & Jacobs, H. T.3 1. University of Dundee, UK.
2.Youngstown State University, USA, 3. Institute of Medical Technology, Tampere, Finland.

    Mitochondrial DNA replicates autonomously from nuclear DNA, but is wholly dependedt on nuclear gnes for its repkication.  Replication of mtDNA is beleived to occur slowly throughout much, if not all of the cell cycle.  As the mtDNA copy number is ordinarily constant in a cell, it must in some sense be cell-cycle regulated.  However, changes in mtDNA copy  number occur naturally at certain stages of development and in particular tissues. It is possible to induce mtDNA amplification by first depleting cells transiently of their mitochondrial DNA, with compounds such as ethidium bromide or deoxycytidine.  Using Brewer-Fangman two-dimensional DNA electrophoresis we have examined mtDNA replication intermediates of untreated human cells, and cells recovereing from transient mtDNA depletion.  These results indicate 1) that there is substantial pausing during human mitochondrial DNA replication and 2) that there is at least two mechanisms of mitochondrial DNA replication in human cultured cells.  One of these mechanisms is upregulated during re-amplification of mtDNA to restore normal copy number.  This mechanism results in the formation of double stranded replication forks, visible as classic Y arcs on 2D gels.  This finding was unexpected as earlier studies have suggested that the replication of the two strands of mammalian mtDNA is intiiated at separate origins, at differnt times, leading to replication intermediates that remain substantially single stranded throughout the replication cycle of mitochondrial DNA.

Meeting:  Nuclear-Mmitochondrial Interactions in Biogenesis & Disease
                    Joint EU Networks Meeting
Date:   19-22 May  1996
Location: Centre Paul Langevin, Aussois, France

MGT1, Recombination Structures, and Inheritance of Mitochondrial DNA in the Yeast Saccharomyces cerevisiae:
Heather E. Lorimer, Bonita J. Brewer, and Walton L. Fangman.
University of Washington, Dept. of Genetics, Seattle, WA,  U.S.A.

     Deletion mutants (rho-) of the mitochondrial genome in Saccharomyces cerevisiae can be preferentially inherited over wild-type mtDNA (rho+) during the clonal development of zygotes.  One class of such mutants (HS rho-) are inherited by  >95% of the diploid cells after matings between HS rho- and rho+ cells.  Another class of mutant mtDNAs (neutral rho-) are seldom inherited in matings with rho+ cells.  HS rho- mtDNAs contain a conserved 300 bp sequence called REP (or ori); neutral rho- mtDNAs lack REP sequences.  Preferential inheritance during the divisions immediately following mating is thought to result from a replication and/or segregation advantage conferred by the high density of REP sequences in HS rho- mtDNAs.
 MGT1 (also known as CCE1), a nuclear gene that is required for the preferential inheritance of HS rho- mtDNA, encodes a recombination junction resolvase.  Analysis of restriction fragments of mitochondrial DNA isolated from mgt1 null mutants reveals an accumulation of singly- and multiply-branched molecules.  We have proposed that heavily cross-linked genomes persist in mgt1 mutants and lead to a decrease in the number of heritable units of mtDNA (Lockshon et al, 1995).  We find that the genomes in rho- mutants are more heavily cross-linked than rho+ genomes and argue that this difference results from an increased opportunity for initiating recombination among the highly-repeated rho- genomes relative to that of lower-copy rho+ genomes.  These results can be interpreted without assigning any specific role of MGT1 in the functioning of REP sequences.  However, we cannot rule out the possibility that the MGT1 resolvase also acts directly on the REP sequence to contribute to the preferential inheritance of HS rho- genomes observed in MGT1 diploids.
     There are variable effects of inactivating the MGT1 gene on the vegetative maintenance of different mtDNAs.  For example, in the absence of MGT1 a 4.6 kb HS rho- (HS8-3) is maintained very poorly, while a 1.8 kb HS rho- deletion derivative of it (HS82d) is maintained relatively well.  Both rho- genomes contain the same REP sequence.  Thus the possibility that the MGT1 resolvase acts specifically through REP sequences during replication or segregation is not supported.  However, these same data indicate that deletion of a sequence restores maintenance in mgt1 mutant cells.  In addition, some rho- mtDNAs lacking REP sequences absolutely require MGT1 for maintenance.  Thus, there is evidence for sequence-specific action of MGT1.  This action may be an activity other than site-specific cleavage of DNA.  We have analyzed the endonuclease activity of MGT1 protein on plasmids containing cloned mtDNAs from HS and neutral rho- cells in vitro.  So far, no sequence specific endonuclease activity on plasmids either containing or lacking REP sequences has been found.
     To further explore the in vivo function of the MGT1 resolvase we have analyzed the native structure of rho- mtDNA by 2-dimensional gel electrophoresis.  Uncut rho- mtDNAs contain complex branched forms, supercoiled, relaxed and nicked circles from unit length through large multimers, double-stranded linears of heterogeneous sizes and heterogeneous, linear single-stranded DNA.  Preliminary evidence indicates that over-expression of MGT1 may result in changes in some of the DNA forms.  Stationary phase cells contain substantially less single-stranded mtDNA than do logarythmically growing cells.  MtDNAs that require MGT1 for maintenance, have similar 2-D gel patterns to mtDNAs that do not require MGT1 for maintenance.

Lockshon, D., S. G. Zweifel, L. L. Freeman-Cook, H. E. Lorimer, B. J. Brewer, and W. L. Fangman. 1995.
A Role for Recombination Junctions in the Segregation of Mitochondrial DNA in Yeast. Cell 81: 1-20.

Meeting: Gordon Research Conference on Mitochondria and Chloroplasts
Date: May 1 - May 6, 1994
Location: Volterra, Italy

Meeting: The Imperial Cancer Research Fund
            Tumour Virus Meeting on Papovaviruses and Adenoviruses
Date:  29th July - 3rd August, 1991
Location: Churchill College, Cambridge, England.

Polyoma and SV40 T Antigens Melt Different Regions Within the Polyoma Replication Origin.
Heather E. Lorimer and Carol Prives, Columbia University, Dept. of Biological Sciences, New York, N.Y.

    The replication origins of Polyomavirus (Py) and SV40 both contain a central palindrome containing four T antigen consensus pentanucleotide (GA/GGGC) binding sites adjacent  to an A/T rich region on the late side.  On the early side of the core origin the two viruses share little sequence homology.  SV40 T antigen was previously shown to induce localized melting in the SV40 core origin (1,2). Alterations in the DNA structure of Py core origin DNA induced by Py T antigen have not been reported.  Furthermore, although both SV40 and Py T antigens bind specifically to each other's origins, they are not capable of supporting replication of DNA containing the reciprocal origin.  We have therfor examined structural changes in Py and SV40 DNA induced by Py and SV40 T antigens using KMnO4 modification.
    Py T antigen exposed Py origin DNA to KMnO4 modification of T residues on the early side of the central palindrome at two A/T regions, one within the core origin and one more distal, located within sites B and C.  Binding of Py DNA to Py T antigen alsoresulted in modification of C residues within the central palindrome.  While SV40 also induced KMnO4 modification site within the Py origin, they differed markedly both qualitatively and quantitatively from those seen  with Py T antigen.  These differences in the structural modifications induced in the Py Origin DNA may explain the failure of SV40 T antigen to mediate replication of Py origin DNA in permissive cells.

(1) Borowiec, J.A., and Hurwitz, J. (1988)  EMBO J.  7: 3149
(2) Parsons, R.,Anderson, M. E., and Tegtmeyer, P. J. Virol. 64: 509