Student-Scientist Curriculum: Integrating Inquiry-Based Research Experiences and Professional Development Activities into an Introductory Biology Laboratory Course.

We designed a 16-week scaffolded student-scientist curriculum using inquiry-based research experiences integrated with professional development activities. This curriculum was implemented to teach undergraduate students enrolled in an introduction to biology course about enzyme activity, biochemical reactions, and alcohol fermentation. While working through the curriculum, students completed the entire scientific process by planning experiments, maintaining laboratory journals, analyzing and interpreting data, peer-reviewing research proposals, and producing and presenting a poster. The overall outcome was for students to complete a multiweek, collaborative, student-scientist project using Saccharomyces cerevisiae as the model organism. Student learning outcomes were evaluated using formative assessments (post-Research on the Integrated Science Curriculum survey and peer- and self-reflection worksheets) and summative assessments (pre/post assessments and assignment grades). Results indicated that more than 50% of the students scored 70% or higher on the collaborative student-scientist project, demonstrated several self-reported learning gains in scientific concepts and skills, and reported they would recommend this laboratory course to their peers. By providing the opportunity for students to carry out the entire scientific process, this curriculum enhanced their technical, analytical, and communication skills.

Zfp36l3, a rodent X chromosome gene encoding a placenta-specific member of the Tristetraprolin family of CCCH tandem zinc finger proteins.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins can bind directly to AU-rich elements (ARE) in mRNA, causing deadenylation and destabilization of the transcripts to which they bind. We describe here a novel fourth mammalian member of the TTP protein family, designated ZFP36L3, which could also bind directly to ARE-containing RNAs and could promote the deadenylation and degradation of ARE-containing target RNAs. Zfp36l3 transcript expression was detected only in placenta and extraembryonic tissues in the mouse. It was expressed throughout development in the placenta and was particularly highly expressed in the cells of the labyrinthine layer of the trophoblastic placenta. Unlike the other family members, the expression of a ZFP36L3-green fluorescent protein fusion protein was entirely cytoplasmic when expressed in 293 cells, even in the presence of the CRM1-dependent nuclear export inhibitor leptomycin B. Zfp36l3 was located on the mouse X chromosome; a similar predicted gene was present on the rat X chromosome, but there was no evidence for a similar gene in humans. ZFP36L3 may thus be a rodent-specific or even murine-specific member of the TTP protein family. Its presumed role in placental physiology may be unique to rodents or murine rodents, but this role may be subsumed by other family members in nonrodents.

The CCCH tandem zinc-finger protein Zfp36l2 is crucial for female fertility and early embryonic development.

The CCCH tandem zinc finger protein, Zfp36l2, like its better-known relative tristetraprolin (TTP), can decrease the stability of AU-rich element-containing transcripts in cell transfection studies; however, its physiological importance is unknown. We disrupted Zfp36l2 in mice, resulting in decreased expression of a truncated protein in which the N-terminal 29 amino acids had been deleted (DeltaN-Zfp36l2). Mice derived from different clones of ES cells exhibited complete female infertility, despite evidence from embryo and ovary transplantation experiments that they could gestate and rear wild-type young. DeltaN-Zfp36l2 females apparently cycled and ovulated normally, and their ova could be fertilized; however, the embryos did not progress beyond the two-cell stage of development. These mice represent a specific model of disruption of the earliest stages of embryogenesis, implicating Zfp36l2, a probable mRNA-binding and destabilizing protein, in the physiological control of female fertility at the level of early embryonic development. This newly identified biological role for Zfp36l2 may have implications for maternal mRNA turnover in normal embryogenesis, and conceivably could be involved in some cases of unexplained human female infertility.

Chorioallantoic fusion defects and embryonic lethality resulting from disruption of Zfp36L1, a gene encoding a CCCH tandem zinc finger protein of the Tristetraprolin family.

The mouse gene Zfp36L1 encodes zinc finger protein 36-like 1 (Zfp36L1), a member of the tristetraprolin (TTP) family of tandem CCCH finger proteins. TTP can bind to AU-rich elements within the 3'-untranslated regions of the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to accelerated mRNA degradation. TTP knockout mice exhibit an inflammatory phenotype that is largely due to increased TNF secretion. Zfp36L1 has activities similar to those of TTP in cellular RNA destabilization assays and in cell-free RNA binding and deadenylation assays, suggesting that it may play roles similar to those of TTP in mammalian physiology. To address this question we disrupted Zfp36L1 in mice. All knockout embryos died in utero, most by approximately embryonic day 11 (E11). Failure of chorioallantoic fusion occurred in about two-thirds of cases. Even when fusion occurred, by E10.5 the affected placentas exhibited decreased cell division and relative atrophy of the trophoblast layers. Although knockout embryos exhibited neural tube abnormalities and increased apoptosis within the neural tube and also generalized runting, these and other findings may have been due to deficient placental function. Embryonic expression of Zfp36L1 at E8.0 was greatest in the allantois, consistent with a potential role in chorioallantoic fusion. Fibroblasts derived from knockout embryos had apparently normal levels of fully polyadenylated compared to deadenylated GM-CSF mRNA and normal rates of turnover of this mRNA species, both sensitive markers of TTP deficiency in cells. We postulate that lack of Zfp36L1 expression during mid-gestation results in the abnormal stabilization of one or more mRNAs whose encoded proteins lead directly or indirectly to abnormal placentation and fetal death.

Polymorphisms in the genes encoding members of the tristetraprolin family of human tandem CCCH zinc finger proteins.

The three known mammalian CCCH tandem zinc finger proteins of the tristetraprolin (TTP) class have recently been demonstrated to be mRNA-binding proteins. The prototype, TTP, functions in normal physiology to promote the instability of the tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor mRNAs. Conversely, these mRNAs are stabilized in TTP-deficient mice, leading to an inflammatory phenotype characterized by overproduction of these cytokines. To explore sequence variations in TTP and its two related proteins, we sequenced genomic DNA encoding the TTP protein (ZFP36) and those of its two known mammalian relatives, ZFP36L1 and ZFP36L2, from 72 to 92 anonymous human subjects from various geographical and ethnic backgrounds. We also sequenced ZFP36 in genomic DNA from 92 subjects exhibiting evidence of excessive TNFalpha action. The resequencing strategy identified 13 polymorphisms in the protein-coding regions of these three genes, of which six would result in amino acid changes; other putative polymorphisms were identified by EST searches. One mutation in ZFP36L1 was a dinucleotide substitution that would prevent splicing of the single intron. This mutation was identified in only one allele of the original 144 sequenced from an adult female Aka Pygmy from the Central African Republic; a second individual with the same variant allele was found by genotyping 58 additional Aka DNA samples. Analysis of mRNA from one of these subject's lymphoblasts confirmed that ZFP36L1 mRNA levels were approximately 50% of those in a comparable sample without the mutation. The functional significance of this and the other polymorphisms identified remains to be determined by both biochemical and population linkage studies.

Members of the tristetraprolin family of tandem CCCH zinc finger proteins exhibit CRM1-dependent nucleocytoplasmic shuttling.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to certain types of AU-rich elements (AREs) in mRNA. Experiments in TTP-deficient mice have shown that TTP is involved in the physiological destabilization of at least two cytokine mRNAs, those encoding tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The two other known mammalian members of the TTP family, CMG1 and TIS11D, also contain ARE-binding CCCH tandem zinc finger domains and can also destabilize ARE-containing mRNAs. To investigate the effects of primary sequence on the subcellular localization of these proteins, we constructed green fluorescent protein fusions with TTP, CMG1, and TIS11D; these were predominantly cytoplasmic when expressed in 293 or HeLa cells. Deletion and mutation analyses revealed functional nuclear export signals in the amino terminus of TTP and in the carboxyl termini of CMG1 and TIS11D. This type of leucine-rich nuclear export signal interacts with the nuclear export receptor CRM1; abrogation of CRM1 activity resulted in nuclear accumulation of TTP, CMG1, and TIS11D. These proteins are thus nucleocytoplasmic shuttling proteins and rely on CRM1 for their export from the nucleus. Although TTP, CMG1, and TIS11D lack known nuclear import sequences, mapping experiments revealed that their nuclear accumulation required an intact tandem zinc finger domain but did not require RNA binding ability. These findings suggest possible roles for nuclear import and export in the regulation of cellular TTP, CMG1, and TIS11D activity.