1978 — 1980 |
Herrick, Glenn |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Equipment For Studying Oxytricha Gene and Genome Structure and Function @ University of Utah College of Medicine |
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1985 — 1991 |
Herrick, Glenn A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Somatic Genome Alterations in Oxytricha
O. fallax contains in its one cell both a micronucleus (MIC) and a macronucleus (MAC). The MAC develops from a mitotis sister of the MIC in the genetically new individual. Once equipped with its new MAC the cell grows clonally. The MAC provides most or all RNA needed for clonal life. The MAC DNA genome consists of about 1000 copies each of 20,000 different, short linear duplex DNAs and carries only 10% of the germline (MIC) sequence complexity. As expected, analyses of cloned MIC DNA regions carrying MAC-destined sequences show MIC-limited sequences between blocks of sequences destined to be on different MAC pieces (inter-MAC). Such analyses also show MIC-limited sequences separating blocks of MAC-destined sequences which are contiguous on the same piece of MAC DNA (intra-MAC), analogous to introns or intervening sequences embedded in genes; this is not a rare phenomenon but has been seen at every MAC-homologous locus we have examined. We will study the sequence rearrangements (involving inter- and intra-MAC sequences) which give rise to separate MAC pieces lacking intervening MIC-limited sequences. All MAC pieces carry dC4A4C4A4C4 at both termini, which we now view as telomeres: this sequence is found at MIC chromosome telomeres as well as internally in MIC DNA at boundaries of inter-MAC blocks; ciliate MAC termini are known to act as telomeres in yeast. To learn how MAC telomeres are generated we will further study inter-MAC boundaries, as well as more fully characterize both MIC and MAC telomere structures. Many MAC pieces belong to small families which appear to arise as products of alternate processing of single MIC loci. We will more fully characterize this phenomenon and perform studies to learn what role such families play. We have characterized a MAC DNA piece, the ploidy of which is modulated during MAC development. What is the role of this DNA and how can the modulated during MAC development. What is the role of this DNA and how can the modulation be manipulated? Several fundamental characteristics of O. fallax practical biology, of MAC development, and of MIC and MAC DNA will be determined. Inter-MAC and intra-MAC related genome rearrangements which we observe as a matter of course seem strongly relevant to genome rearrangements controlling the immune system and to oncogene-related Robertsonian rearrangements in cancer etiology (telomeres dictate the rules of Robertsonian rearrangments). We argue that somatic genome rearrangements may yet be discovered in a wide variety of developmental paths. Alternate juxtapositioning of sequences likely plays a major role in the control of gene expression in man and his viruses.
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0.958 |
1992 — 1994 |
Herrick, Glenn A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Somatic Genome Alterations in Oxytricha Fallax |
0.958 |
1997 — 2000 |
Herrick, Glenn A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Somatic Genome Alterations in Oxytrica Fallax
DESCRIPTION: The hypotricous ciliates are remarkable organisms. Like all ciliates they have two nuclei; a transcriptionally silent micronucleus and a transcriptionally active macronucleus (nuclei). The DNA of the micronucleus is transmitted sexually to the next generation and in mitotic cell divisions by standard eukaryotic mechanisms. The DNA of the macronucleus is found in gene-sized pieces that amplified, is not transmitted sexually to the next generation, only a fraction of the micronuclear DNA (`10%) is present and the gene-sized pieces are segregated amitotically in the mitotic cell divisions. In addition, the nuclear genome uses TAA and TAG, which are normally stop codons, as glutamine codons. After conjugation, the DNA destined to become the macronucleus is amplified in polytene chromosomes. There is chromosome breakage, loss of large portions of the DNA and new telomeres are made and added on to the gene-sized pieces that are left. The sequences that are lost are referred to as internally eliminated sequences or IESs. So this nucleus is genetically a dead-end. It will not be transmitted to the next generation. IESs fall both in genes and between genes. Especially in the many examples of IESs that fall in the coding region, the excision of the IES must be exact. The starting point for these studies was the locus designated 81. It has five short IESs and three TBE1 elements. The TBE1 element is a 4.1 kb transposon that is present in the micronuclear genome in about 2,200 copies per haploid genome. All of these sequences are eliminated in the production of the macronucleus. They are flanked by AAT target duplications. There are also short IESs and there may be 105 short IESs in the genome. TBE1 has five long open reading frames. At least two of these are conserved in the four TBE1 elements that have been sequenced. The 57 kD ORF shows homology to protein kinases and zinc fingers and the 42 kD ORF shows homology to transposases, which include mariner. TBE2 and TBE3 have also been sequenced. They share about 40% sequence identity. When TBE1 excises, it does not appear to use gap-repair and it generates circular molecules. The RNA for the TBE1 elements is very labile, heterogeneous, and does not appear to be polyadenylated. Nested PCR and probing gave a signal, so there may a small fraction that is polyadenylated. Bulk PCR sequencing using a set of primers suggest that the 1,500 to 3,000 TBE1 have both primers and they are at a fairly canonical distance apart. Dr. Herrick's proposal seeks to understand how these the sequence similarity is maintained among the TBE1 elements. Population dynamics and evolution will be studied to distinguish between different selection models. Secondly, the developmentally regulated excision of TBE1 will be examined. Long term goals include developing an in vitro system and attempting to understand the coevolution of TBEs and their hosts.
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0.958 |
2001 — 2002 |
Herrick, Glenn |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Telomere Formation, Sites and Intermediates, During Macronuclear Development of Oxytricha Trifallax
A new technique allows rapid and precise mapping of telomere addition sites (TASs) at chromosome ends of the hypotrichous ciliate macronucleus. The macronucleus is carved developmentally from a copy of the cell's gremlin nucleus, involving the generation of >40,000 new telomeres, following the breakage of chromatids of polytenized germline chromosomes, creating chromosomes with just one or a few genes each. Some chromosomes are created using exactly the same TAS pair (called "fixed TASs"), but others are generated by alternative processing. In this case, some chromatids escape breakage altogether and use a heterogeneous set of TASs in a region dictated by cis-acting sequences (called "mixed" TASs). One hypothesis of how this may work is the "hesitant cutter" hypothesis, whereby the TAS spectrum results from the action of a loose complex of the chromatid cutter and enzyme, telomerase. This model suggests that the region is anchored by a binding site ("CBS") for the complex and a relatively poor cut site, such that the cutter is slow to cut; meanwhile the telomerase dissociates, the cut is made, the end left unprotected to nuclease erosion. The nuclease could then proceed through the 3' strand until it pauses under the influence of a cis-acting pause site, often at dT in a string of dTs. This could allow telomerase to act when the erosion is paused. Mapping TASs in mixed regions will allow the uncovering of three types of cis-acting sequences: the CBS, cut, and pause sites. Numerous allelic variants of TAS regions will be so mapped, refining the effective site sequences. In addition, fixed TASs will be mapped to allow the refinement of a consensus site already identified, 3' TAY (Y=purine). This project will help illuminate the processes of chromosome breakage, telomere formation and the action of telomerase. Basic understanding of these processes will be key to understanding cellular senescence.
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