Biomedical subjects

Anthony L Gotter

Publications and source records attributed to Anthony L Gotter.

7 recordsLinked to original sources

PubMed2006

Mammalian TIMELESS and Tipin are evolutionarily conserved replication fork-associated factors.

The function of the mammalian TIMELESS protein (TIM) has been enigmatic. TIM is essential for early embryonic development, but little is known regarding its biochemical and cellular function. Although identified based on similarity to a Drosophila circadian clock factor, it also shares similarity with a second family of proteins that is more widely conserved throughout eukaryotes. Members of this second protein family in yeast (S.c. Tof1p, S.p. Swi1p) have been implicated in DNA synthesis, S-phase-dependent checkpoint activation and chromosome cohesion, three processes coordinated at the level of the replication fork complex. The present work demonstrates that mammalian TIM and its constitutive binding partner, Tipin (ortholog of S.c. Csm3p, S.p. Swi3p), are replisome-associated proteins. Both proteins associate with components of the endogenous replication fork complex, and are present at BrdU-positive DNA replication sites. Knock-down of TIM also compromises DNA replication efficiency. Further, the direct binding of the TIM-Tipin complex to the 34 kDa subunit of replication protein A provides a biochemical explanation for the potential coupling role of these proteins. Like TIM, Tipin is also involved in the molecular mechanism of UV-dependent checkpoint activation and cell growth arrest. Tipin additionally associates with peroxiredoxin2 and appears to be involved in checkpoint responses to H(2)O(2), a role recently described for yeast versions of TIM and Tipin. Together, this work establishes TIM and Tipin as functional orthologs of their replisome-associated yeast counterparts capable of coordinating replication with genotoxic stress responses, and distinguishes mammalian TIM from the circadian-specific paralogs from which it was originally identified.

Animals↗

PubMed2006

A Timeless debate: resolving TIM's noncircadian roles with possible clock function.

Mammalian TIMELESS (TIM) was identified due to its sequence similarity to Drosophila TIM, an essential circadian clock protein in flies. Published literature is inconsistent regarding the rhythmic expression of mammalian Tim, the interaction of the TIM protein with other clock proteins and its role in regulating clock gene transcription. Comprehensive sequence analysis not only demonstrates that mammalian TIM is more similar to a second, TIM-like sequence in Drosophila (TIMEOUT), but is also a member of an evolutionarily conserved family of TIM orthologs that is distinct from the circadian-specific TIM proteins found in insects. The vital cellular function of these widely conserved TIM orthologs makes it difficult to determine the specific role of mammalian TIM in the circadian clock mechanism.

Animals↗

PubMed2003

A palindrome-mediated mechanism distinguishes translocations involving LCR-B of chromosome 22q11.2.

Two known recurrent constitutional translocations, t(11;22) and t(17;22), as well as a non-recurrent t(4;22), display derivative chromosomes that have joined to a common site within the low copy repeat B (LCR-B) region of 22q11.2. This breakpoint is located between two AT-rich inverted repeats that form a nearly perfect palindrome. Breakpoints within the 11q23, 17q11 and 4q35 partner chromosomes also fall near the center of palindromic sequences. In the present work the breakpoints of a fourth translocation involving LCR-B, a balanced ependymoma-associated t(1;22), were characterized not only to localize this junction relative to known genes, but also to further understand the mechanism underlying these rearrangements. FISH mapping was used to localize the 22q11.2 breakpoint to LCR-B and the 1p21 breakpoint to single BAC clones. STS mapping narrowed the 1p21.2 breakpoint to a 1990 bp AT-rich region, and junction fragments were amplified by nested PCR. Junction fragment-derived sequence indicates that the 1p21.2 breakpoint splits a 278 nt palindrome capable of forming stem-loop secondary structure. In contrast, the 1p21.2 reference genomic sequence from clones in the database does not exhibit this configuration, suggesting a predisposition for regional genomic instability perhaps etiologic for this rearrangement. Given its similarity to known chromosomal fragile site (FRA) sequences, this polymorphic 1p21.2 sequence may represent one of the FRA1 loci. Comparative analysis of the secondary structure of sequences surrounding translocation breakpoints that involve LCR-B with those not involving this region indicate a unique ability of the former to form stem-loop structures. The relative likelihood of forming these configurations appears to be related to the rate of translocation occurrence. Further analysis suggests that constitutional translocations in general occur between sequences of similar melting temperature and propensity for secondary structure.

Base Sequence↗

PubMed2003

A novel sequence-based approach to localize translocation breakpoints identifies the molecular basis of a t(4;22).

Low copy repeats (LCRs) located in 22q11.2, especially LCR-B, are susceptible to rearrangements associated with several relatively common constitutional disorders. These include DiGeorge syndrome, Velocardiofacial syndrome, Cat-eye syndrome and recurrent translocations of 22q11 including the constitutional t(11;22) and t(17;22). The presence of palindromic AT-rich repeats (PATRRs) within LCR-B of 22q11.2, as well as within the 11q23 and 17q11 regions, has suggested a palindrome-mediated, stem-loop mechanism for the generation of such recurring constitutional 22q11.2 translocations. The mechanism responsible for non-recurrent 22q11.2 rearrangements is presently unknown due to the extensive effort required for breakpoint cloning. Thus, we have developed a novel fluorescence in-situ hybridization and primed in-situ hybridization (PRINS) approach and rapidly localized the breakpoint of a non-recurrent 22q11.2 translocation, a t(4;22). Multiple primer pairs were designed from the sequence of a 200 kb, chromosome 4, breakpoint-spanning BAC to generate PRINS probes. Amplification of adjacent primer pairs, labeled in two colors, allowed us to narrow the 4q35.1 breakpoint to a 6.7 kb clonable region. Application of our improved PRINS protocol facilitated fine-mapping the translocation breakpoints within 4q35.1 and 22q11.2, and permitted rapid cloning and analysis of translocation junction fragments. To confirm the PRINS localization results, PCR mapping of t(4;22) somatic cell hybrid DNA was employed. Analysis of the breakpoints demonstrates the presence of a 554 bp palindromic sequence at the chromosome 4 breakpoint and a 22q11.2 location within the same PATRR as the recurrent t(11;22) and t(17;22). The sequence of this breakpoint further suggests that a stem-loop secondary structure mechanism is responsible for the formation of other, non-recurrent translocations involving LCR-B of 22q11.2.

AT Rich Sequence↗

PubMed2003

Tipin, a novel timeless-interacting protein, is developmentally co-expressed with timeless and disrupts its self-association.

The mouse Timeless gene (mTim) was identified originally on the basis of its similarity to a Drosophila circadian gene, but has no substantiated role in the circadian clock mechanism. The importance of mTim in cellular processes involved in development, however, is undeniable, since targeted mutagenesis of this gene arrests embryonic development. To connect mTim to known pathways controlling cellular processes important for early development, a yeast two-hybrid approach was used to identify embryonic mTIM-interacting proteins. One positive interactor, a previously uncharacterized protein that is here termed TIPIN (TIMELESS interacting protein), was shown to interact with mTIM in vitro and in cultured cells. mTim and Tipin transcripts are co-expressed in similar tissues during embryonic development and in the adult brain. In transiently transfected cultures, mTIM promotes the nuclear localization of TIPIN. Immunoprecipitation experiments suggest that TIPIN is capable of regulating mTIM activity by disrupting the ability of mTIM to form homo-multimeric complexes. Together, these results indicate that mTIM forms a functional complex with TIPIN, and provide a starting point from which to link mTim to biochemical pathways controlling vital cellular functions.

3T3 Cells↗

PubMed2003

Constructing a feedback loop with circadian clock molecules from the silkmoth, Antheraea pernyi.

Circadian clocks are important regulators of behavior and physiology. The circadian clock of Drosophila depends on an autoinhibitory feedback loop involving dCLOCK, CYCLE (also called dBMAL, for Drosophila brain and muscle ARNT-like protein), dPERIOD, and dTIMELESS. Recent studies suggest that the clock mechanism in other insect species may differ strikingly from that of Drosophila. We cloned Clock, Bmal, and Timeless homologs (apClock, apBmal, and apTimeless) from the silkmoth Antheraea pernyi, from which a Period homolog (apPeriod) has already been cloned. In Schneider 2 (S2) cell culture assays, apCLOCK:apBMAL activates transcription through an E-box enhancer element found in the 5' region of the apPeriod gene. Furthermore, apPERIOD can robustly inhibit apCLOCK: apBMAL-mediated transactivation, and apTIMELESS can augment this inhibition. Thus, a complete feedback loop, resembling that found in Drosophila, can be constructed from silkmoth CLOCK, BMAL, PERIOD, and TIMELESS. Our results suggest that the circadian autoinhibitory feedback loop discovered in Drosophila is likely to be widespread among insects. However, whereas the transactivation domain in Drosophila lies in the C terminus of dCLOCK, in A. pernyi, it lies in the C terminus of apBMAL, which is highly conserved with the C termini of BMALs in other insects (except Drosophila) and in vertebrates. Our analysis sheds light on the molecular function and evolution of clock genes in the animal kingdom.

ARNTL Transcription Factors↗

PubMed2003

Illuminating the circadian clock in monarch butterfly migration.

Migratory monarch butterflies use a time-compensated Sun compass to navigate to their overwintering grounds in Mexico. Here, we report that constant light, which disrupts circadian clock function at both the behavioral and molecular levels in monarchs, also disrupts the time-compensated component of flight navigation. We further show that ultraviolet light is important for flight navigation but is not required for photic entrainment of circadian rhythms. Tracing these distinct light-input pathways into the brain should aid our understanding of the clock-compass mechanisms necessary for successful migration.

Animal Migration↗