TEMPORAL ORGANIZATION OF FEEDING IN SYRIAN HAMSTERS WITH A GENETICALLY ALTERED CIRCADIAN PERIOD

The variation in spontaneous meal patterning was studied in three genotypes (tau +/+, tau +/?and tau ?/?) of the Syrian hamster with an altered circadian period. Feeding activity was monitored continuously in 13 individuals from each genotype in constant dim light conditions. All three genotypes had on average six feeding episodes during the circadian cycle (about 20h in homozygous tau mutants and 22h in heterozygotes compared with 24h in wild-type hamsters). Thus, homozygous tau mutant hamsters had significantly more feeding episodes per 24h than wild types, and heterozygotes were intermediate. The average duration of feeding bouts (FBs) was indistinguishable (around 30 minutes) among the three genotypes, whereas the intermeal (IM) intervals were significantly shorter for homozygote tau mutant hamsters (99 minutes), intermediate for heterozygotes (116 minutes), and the longest for wild-type hamsters (148 minutes). Thus, the meal-to-meal duration was on average 25% shorter in homozygous tau mutants (16% in heterozygous) than in wild-type hamsters. The reduction of the circadian period has a pronounced effect on short-term feeding rhythms and meal frequency in hamsters carrying the tau mutation. (Chronobiology International, 18(4), 657–664, 2001)     

Genetics of Circadian Clocks

The isolation of circadian clock mutants in Neurospora crassa and Drosophila melanogaster have identified numerous genes whose function is necessary for the normal operation of the circadian clock. In Neurospora many of these mutants map to a single locus called frq, whose properties suggest that its gene product is intimately involved in clock function. In Drosophila mutations at the per locus also suggest a significant role for the product of this gene in the insect clock mechanism. The per gene has been cloned and its gene product identified as a proteoglycan, most likely a membrane protein involved in affecting the ionic or electrical properties of cells in which it is located. Future progress in elucidating the mechanisms of circadian clocks are likely to come from continued analysis of clock mutants, both at the genetic and molecular levels.     

Clock Genes Influence Gene Expression in Growth Plate and Endochondral Ossification in Mice

We have previously shown transient promotion by parathyroid hormone of Period-1 (Per1) expression in cultured chondrocytes. Here we show the modulation by clock genes of chondrogenic differentiation through gene transactivation of the master regulator of chondrogenesis Indian hedgehog (IHH) in chondrocytes of the growth plate. Several clock genes were expressed with oscillatory rhythmicity in cultured chondrocytes and rib growth plate in mice, whereas chondrogenesis was markedly inhibited in stable transfectants of Per1 in chondrocytic ATDC5 cells and in rib growth plate chondrocytes from mice deficient of brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1). Ihh promoter activity was regulated by different clock gene products, with clear circadian rhythmicity in expression profiles of Ihh in the growth plate. In BMAL1-null mice, a predominant decrease was seen in Ihh expression in the growth plate with a smaller body size than in wild-type mice. BMAL1 deficit led to disruption of the rhythmic expression profiles of both Per1 and Ihh in the growth plate. A clear rhythmicity was seen with Ihh expression in ATDC5 cells exposed to dexamethasone. In young mice defective of BMAL1 exclusively in chondrocytes, similar abnormalities were found in bone growth and Ihh expression. These results suggest that endochondral ossification is under the regulation of particular clock gene products expressed in chondrocytes during postnatal skeletogenesis through a mechanism relevant to the rhythmic Ihh expression.     

Structure of the frequency‐interacting RNA helicase: a protein interaction hub for the circadian clock

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ‐interacting RNA Helicase (FRH) rhythmically represses gene expression by the white‐collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ‐interacting region at the FRH N‐terminus has variable structure in the absence of FRQ. A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ. A V142G substitution near the N‐terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N‐terminus and KOW module.     

The circadian system of c-fos deficient mice

We examined the role of c-fos in the synchronization of circadian rhythms to environmental light cycles using a line of gene-targeted mice carrying a null mutation at this locus. Circadian locomotor rhythms in mutants had similar periods as wild-type controls but took significantly longer than controls to entrain to 12:12 light-dark cycles. Light-induced phase shifts of rhythms in constant dark were attenuated in mutants although the circadian timing of phase delays and advances was not changed. A functional retinohypothalamic projection was indicated from behavioral results and light-induced jun-B expression in the SCN. The results indicate that while c-fos activation is not an absolute requirement for rhythm generation nor photic responses, it is required for normal entrainment of the mammalian biological clock.Abbreviations SCN suprachiasmatic nucleus - RHT retinohypothalamic tract - IEG immediate early genes - NGF nerve growth factor - VIP vasoactive intestinal polypeptide - DD constant darkness - CT circadian time     

Genetic knockouts and knockins in human somatic cells

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.     

Targeted knockout and <Emphasis Type="Italic">lacZ</Emphasis> reporter expression of the mouse <Emphasis Type="Italic">Tmhs</Emphasis> deafness gene and characterization of the <Emphasis Type="Italic">hscy-2J</Emphasis> mutation

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ^tm1Kjn ) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of β-galactosidase activity in Tmhs ^tm1Kjn heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.     

The Luteinizing Hormone Surge Regulates Circadian Clock Gene Expression in the Chicken Ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。     

The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.)

Summary A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%–3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about I%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels.     

Mammalian cryptochromes impinge on cell cycle progression in a circadian clock-independent manner

By gating cell cycle progression to specific times of the day, the intracellular circadian clock is thought to reduce the exposure of replicating cells to potentially hazardous environmental and endogenous genotoxic compounds. Although core clock gene defects that eradicate circadian rhythmicity can cause an altered in vivo genotoxic stress response and aberrant proliferation rate, it remains to be determined to what extent these cell cycle related phenotypes are due to a cell-autonomous lack of circadian oscillations. We investigated the DNA damage sensitivity and proliferative capacity of cultured primary Cry1^?/-?|Cry2^?/- fibroblasts. Contrasting previous in vivo studies, we show that the absence of CRY proteins does not affect the cell-autonomous DNA damage response upon exposure of primary cells in vitro to genotoxic agents, but causes cells to proliferate faster. By comparing primary wild-type, Cry1^?/-?|Cry2^?/-, Cry1^+/-|Cry2^-/- and Cry1^-/-|Cry2^+/- fibroblasts, we provide evidence that CRY proteins influence cell cycle progression in a cell-autonomous, but circadian clock-independent manner and that the accelerated cell cycle progression of Cry-deficient cells is caused by global dysregulation of Bmal1-dependent gene expression. These results suggest that the inconsistency between in vivo and in vitro observations might be attributed to systemic circadian control rather than a direct cell-autonomous control.     

Gonadotropic regulation of circadian clockwork in rat granulosa cells

The circadian clock is responsible for the generation of circadian rhythms in hormonal secretion and metabolism. These peripheral clocks could be reset by various cues in order to adapt to environmental variations. The ovary can be characterized as having highly dynamic physiology regulated by gonadotropins. Here, we aimed to address the status of circadian clock in the ovary, and to explore how gonadotropins could regulate clockwork in granulosa cells (GCs). To this end, we mainly utilized the immunohistochemistry, RT-PCR, and real-time monitoring of gene expression methods. PER1 protein was constantly abundant across the daily cycle in the GCs of immature ovaries. In contrast, PER1 protein level was obviously cyclic through the circadian cycle in the luteal cells of pubertal ovaries. In addition, both FSH and LH induced Per1 expression in cultured immature and mature GCs, respectively. The promoter analysis revealed that the Per1 expression was mediated by the cAMP response element binding protein. In cultured transgenic GCs, both FSH and LH also induced the circadian oscillation of Per2. However, the Per2 oscillation promoted by FSH quickly dampened within only one cycle, whereas the Per2 oscillation promoted by LH was persistently maintained. Collectively, these findings strongly suggest that both FSH and LH play an important role in regulating circadian clock in the ovary; however, they might exert differential actions on the clockwork in vivo due to each specific role within ovarian physiology.