Molecular Genetic Programs Research Articles

AP-2α and AP-2β cooperatively function in the craniofacial surface ectoderm to regulate chromatin and gene expression dynamics during facial development.

The facial surface ectoderm is essential for normal development of the underlying cranial neural crest cell populations, providing signals that direct appropriate growth, patterning, and morphogenesis. Despite the importance of the ectoderm as a signaling center, the molecular cues and genetic programs implemented within this tissue are understudied. Here, we show that removal of two members of the AP-2 transcription factor family, AP-2α and AP-2ß, within the early embryonic ectoderm of the mouse leads to major alterations in the craniofacial complex. Significantly, there are clefts in both the upper face and mandible, accompanied by fusion of the upper and lower jaws in the hinge region. Comparison of ATAC-seq and RNA-seq analyses between controls and mutants revealed significant changes in chromatin accessibility and gene expression centered on multiple AP-2 binding motifs associated with enhancer elements within these ectodermal lineages. In particular, loss of these AP-2 proteins affects both skin differentiation as well as multiple signaling pathways, most notably the WNT pathway. We also determined that the mutant clefting phenotypes that correlated with reduced WNT signaling could be rescued by Wnt1 ligand overexpression in the ectoderm. Collectively, these findings highlight a conserved ancestral function for AP-2 transcription factors in ectodermal development and signaling, and provide a framework from which to understand the gene regulatory network operating within this tissue that directs vertebrate craniofacial development.

Perceived learning environment and academic motivation: An explanatory mixed methods study in a Molecular Biology and Genetics program.

Lack of motivation of students is a major concern for the instructors and administration in universities, and it is in their interest to address these issues in the most effective and efficient way. The purpose of this explanatory sequential mixed-method study is to examine the predictive power of the learning environment on student academic motivation and to learn students' opinions on this issue. One hundred twenty students enrolled at the Molecular Biology and Genetics program completed the Academic Motivation and Learning Environment Perception scales, Quantitative findings indicated that only the activities dimension of the learning environment has a predictive power on academic motivation. Focus group interviews conducted with 16 students validated the quantitative findings. Students particularly drew attention to activities that foster active participation in the learning process, emphasizing the vitality of being engaged and of having real-life experiences in their learning environment.

Межштаммовая гибридизация дрожжей Kluyveromyces lactis для создания штаммов, активно сбраживающих лактозу

A molecular genetic study of Kluyveromyces lactis yeasts isolated from various dairy products in the countries of the former Soviet Union and other regions of the world has been carried out. Based on physiological tests, four strains were selected that carry different LAC loci and are characterized by good fermentation intensity: VKM Y-1339 (LAC3), VKM Y-1333 (LAC3), NRRL Y-1118 (LAC1), and NRRL Y-1140 (LAC2). Eleven hybrids of the selected strains with different rates of lactose fermentation were obtained. No correlation was found between the intensity of lactose fermentation and the amino acid sequences of the LAC12 lactose permease gene of the LAC1, LAC2, and LAC3 loci. Apparently, a specific combination of genotypes of crossed strains has a more significant effect on the fermentation activity. The results obtained showed that inter-strain hybridization of K. lactis dairy yeast is an effective method for creating new strains with high fermentation capacity. Hybrids H2-3 (NRRL Y-1118 × VKM Y-1333) and H3-3 (NRRL Y-1140 × VKM Y-1333) with the highest ability to ferment lactose are of interest for further molecular genetic research and breeding programs. Key words: Kluyveromyces lactis, β-galactosidase, lactose permease, LAC4, LAC12, LAC1 locus, LAC2 locus, LAC3 locus, inter-strain hybridization, lactose fermentation, heterosis Acknowledgment - The authors are grateful to the Genomic Center of the Kurchatov Institute SRC---GosNIIgentika for sequencing the nucleotide sequences of the LAC12 genes for lactose permease on the Applied Biosystems 3730 automated analyzer. Funding - This work was supported by an internal grant from the National Research Center Kurchatov Institute (order of the National Research Center Kurchatov Institute No. 1779).

Update on the clinical utility of an RNA interference-based treatment: focus on Patisiran.

RNA interference (RNAi) is a naturally existing endogenous mechanism for post-transcriptional gene regulation, nowadays commonly utilized for functional characterization of genes and development of potential treatment strategies for diseases. RNAi-based studies for therapy, after being examined for over a decade, are finally in the pipeline for developing a potential treatment for the mutated transthyretin (TTR) gene, which gives rise to a dysfunctional TTR protein. This dysfunctional protein causes TTR amyloidosis (ATTR), an inherited, progressively incapacitating, and often fatal genetic disorder. TTR is a protein produced in the liver, and functions as a carrier for retinol-binding protein and also thyroxine. This protein facilitates the transport of vitamin A around the human body. A mutation or misprint in the code of this protein results in an abnormal folding of the protein. Therefore, not only does the transportation of the vitamin A become disabled, but also there will be formation of clusters called amyloid deposits, which attack the heart and the nerves causing some patients to be unconditionally bound to bed. ATTR is a hereditary autosomal dominant disease with a 50% chance of inheritance by offspring, even with just one of the parents having a single defective allele of this gene. Alnylam Pharmaceuticals worked on the concept of RNAi therapy for years, which led to the introduction of lipid nanoparticles encircling small interfering RNAs. The drug showed extremely positive results since the first trial, and a great percentage of defective protein reduction. This drug was later named Patisiran.

Diversification of Root Hair Development Genes in Vascular Plants.

The molecular genetic program for root hair development has been studied intensively in Arabidopsis (Arabidopsis thaliana). To understand the extent to which this program might operate in other plants, we conducted a large-scale comparative analysis of root hair development genes from diverse vascular plants, including eudicots, monocots, and a lycophyte. Combining phylogenetics and transcriptomics, we discovered conservation of a core set of root hair genes across all vascular plants, which may derive from an ancient program for unidirectional cell growth coopted for root hair development during vascular plant evolution. Interestingly, we also discovered preferential diversification in the structure and expression of root hair development genes, relative to other root hair- and root-expressed genes, among these species. These differences enabled the definition of sets of genes and gene functions that were acquired or lost in specific lineages during vascular plant evolution. In particular, we found substantial divergence in the structure and expression of genes used for root hair patterning, suggesting that the Arabidopsis transcriptional regulatory mechanism is not shared by other species. To our knowledge, this study provides the first comprehensive view of gene expression in a single plant cell type across multiple species.

Insights into brain development and disease from neurogenetic analyses in Drosophila melanogaster.

Groundbreaking work by Obaid Siddiqi has contributed to the powerful genetic toolkit that is now available for studying the nervous system of Drosophila. Studies carried out in this powerful neurogenetic model system during the last decade now provide insight into the molecular mechanisms that operate in neural stem cells during normal brain development and during abnormal brain tumorigenesis. These studies also provide strong support for the notion that conserved molecular genetic programs act in brain development and disease in insects and mammals including humans.

The role of the cell wall compartment in mutualistic symbioses of plants.

Plants engage in mutualistic interactions with microbes that improve their mineral nutrient supply. The most wide-spread symbiotic association is arbuscular mycorrhiza (AM), in which fungi of the order Glomeromycota invade roots and colonize the cellular lumen of cortical cells. The establishment of this interaction requires a dedicated molecular-genetic program and a cellular machinery of the plant host. This program is partially shared with the root nodule symbiosis (RNS), which involves prokaryotic partners collectively referred to as rhizobia. Both, AM and RNS are endosymbioses that involve intracellular accommodation of the microbial partner in the cells of the plant host. Since plant cells are surrounded by sturdy cell walls, root penetration and cell invasion requires mechanisms to overcome this barrier while maintaining the cytoplasm of the two partners separate during development of the symbiotic association. Here, we discuss the diverse functions of the cell wall compartment in establishment and functioning of plant symbioses with the emphasis on AM and RNS, and we describe the stages of the AM association between the model organisms Petunia hybrida and Rhizophagus irregularis.

Managing mainstream genomics - knowing and showing quality

The RCPA QAP Molecular Genetics Program (in association with the Human Genetics Society of Australasia) provides external quality assurance programs (EQA), quality assessment and educational modules to public and private pathology laboratories in Australasia and further afield. The Program obtained NATA accreditation to the ISO/IEC 17043:2010 Conformity Assessment - General Requirements for Proficiency Testing Standard in 2013. The Program also administers molecular genetics modules from the European Molecular Quality Network, based in the UK, and the CF Network, based in Belgium. A fundamental aim of the Program is to further improve the quality of molecular genetics testing and reporting in Australasia by establishing guidelines with the assistance of key opinion leaders and subject matter experts. A significant focus area for the Molecular Genetics program is the Somatic Cancer Mutation detection module incorporating EGFR, KRAS and BRAF genes. An overview of participants' performance, specifically with respect to genotyping and associated clinical interpretations in this and other Molecular Genetics modules will be presented. Examples of rare disorders and infrequently tested markers such as anaplastic lymphoma kinase (ALK) translocations in non-small cell lung carcinoma (NSCLC) and medulloblastoma, which are being addressed in the form of a sample exchange, will also be discussed.

Alternative Roles for CRISPR/Cas Systems in Bacterial Pathogenesis

1Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University School of Medicine, Atlanta, Georgia, United States ofAmerica, 2Emory Vaccine Center, Emory University, Atlanta, Georgia, United States of America, 3Yerkes National Primate Research Center, Emory University, Atlanta,Georgia, United States of America, 4Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, United States ofAmerica

Bidirectional Notch activation represses fusion competence in swarming adultDrosophilamyoblasts

A major aspect of indirect flight muscle formation during adult Drosophila myogenesis involves transition of a semi-differentiated and proliferating pool of myoblasts to a mature myoblast population, capable of fusing with nascent myotubes and generating mature muscle fibers. Here we examine the molecular genetic programs underlying these two phases of myoblast differentiation. We show that the cell adhesion proteins Dumbfounded (Duf) and Sticks and stones (Sns), together with their paralogs Roughest (Rst) and Hibris (Hbs), respectively, are required for adhesion of migrating myoblasts to myotubes and initiation of myoblast-myotube fusion. As myoblasts approach their myotube targets, they are maintained in a semi-differentiated state by continuous Notch activation, where each myoblast provides the ligand Delta to its neighbors. This unique form of bidirectional Notch activation is achieved by finely tuning the levels of the ligand and receptor. Activation of Notch signaling in myoblasts represses expression of key fusion elements such as Sns. Only upon reaching the vicinity of the myotubes does Notch signaling decay, leading to terminal differentiation of the myoblasts. The ensuing induction of proteins required for fusion enables myoblasts to fuse with the myotubes and give rise to subsequent muscle fiber growth.

Expression and function of the empty spiracles gene in olfactory sense organ development of Drosophila melanogaster

In Drosophila, the cephalic gap gene empty spiracles plays key roles in embryonic patterning of the peripheral and central nervous system. During postembryonic development, it is involved in the development of central olfactory circuitry in the antennal lobe of the adult. However, its possible role in the postembryonic development of peripheral olfactory sense organs has not been investigated. Here, we show that empty spiracles acts in a subset of precursors that generate the olfactory sense organs of the adult antenna. All empty spiracles-expressing precursor cells co-express the proneural gene amos and the early patterning gene lozenge. Moreover, the expression of empty spiracles in these precursor cells is dependent on both amos and lozenge. Functional analysis reveals two distinct roles of empty spiracles in the development of olfactory sense organs. Genetic interaction studies in a lozenge-sensitized background uncover a requirement of empty spiracles in the formation of trichoid and basiconic olfactory sensilla. MARCM-based clonal mutant analysis reveals an additional role during axonal targeting of olfactory sensory neurons to glomeruli within the antennal lobe. Our findings on empty spiracles action in olfactory sense organ development complement previous studies that demonstrate its requirement in olfactory interneurons and, taken together with studies on the murine homologs of empty spiracles, suggest that conserved molecular genetic programs might be responsible for the formation of both peripheral and central olfactory circuitry in insects and mammals.

Hematopoietic stem cells: generation and self-renewal

Adult stem cells hold great promise for future therapeutic applications. Hematopoietic stem cells (HSCs) are among the best-characterized adult stem cells. As such, these cells provide a conceptual framework for the study of adult stem cells from other organs. Here, we review the current knowledge of HSC generation during embryonic development and HSC maintenance in the bone marrow (BM) during adult life. Recent scientific progress has demonstrated that the development of HSCs involves many anatomical sites in the embryo, but the relative contribution of each of these sites to the adult HSC pool remains controversial. Specialized anatomical sites in the BM have been identified as stem cell niches, and these play essential roles in regulating the self-renewal and differentiation of HSCs through recently identified signaling pathways. Extracellular signaling from stem cell niches must integrate with the intracellular molecular machinery and/or genetic programs to regulate HSC fate choice. The exact cellular and/or molecular mechanisms defining stem cell niche and 'stemness' of HSC is largely unknown although substantial progress has been made recently. Hence, many questions remain to be answered even in this relatively well-defined model of stem cell biology.

Long QT and Brugada syndrome gene mutations in New Zealand

Genetic testing in long QT syndrome (LQTS) is moving from research into clinical practice. We have recently piloted a molecular genetics program in a New Zealand research laboratory with a view to establishing a clinical diagnostic service. This study sought to report the spectrum of LQTS and Brugada mutations identified by a pilot LQTS gene testing program in New Zealand. Eighty-four consecutive index cases referred for LQT gene testing, from New Zealand and Australia, were evaluated. The coding sequence and splice sites of 5 LQTS genes (KCNQ1, HERG, SCN5A, KCNE1, and KCNE2) were screened for genomic variants by transgenomics denaturing high-performance liquid chromatography (dHPLC) system and automated DNA sequencing. Forty-five LQTS mutations were identified in 43 patients (52% of the cohort): 25 KCNQ1 mutations (9 novel), 13 HERG mutations (7 novel), and 7 SCN5A mutations (2 novel). Forty patients had LQTS, and 3 had Brugada syndrome. Mutations were identified in 14 patients with resuscitated sudden cardiac death: 4 KCNQ1, 5 HERG, 5 SCN5A. In 17 cases there was a family history of sudden cardiac death in a first-degree relative: 8 KCNQ1, 6 HERG, 2 SCN5A, and 1 case with mutations in both KCNQ1 and HERG. The spectrum of New Zealand LQTS and Brugada mutations is similar to previous studies. The high proportion of novel mutations (40%) dictates a need to confirm pathogenicity for locally prevalent mutations. Careful screening selection criteria, cellular functional analysis of novel mutations, and development of locally relevant control sample cohorts will all be essential to establishing regional diagnostic services.

Obituary: Lorraine A. Flaherty (1946–2006)

On February 22, 2006, Lorraine (Lori) A. Flaherty, Ph.D., lost her long battle with cancer. As was her wish, she died at home with her family (husband Joe and children Joey and Amy) and close friends by her side. Born in Rochester, NY, on January 8, 1946, Lori received her B.S. in Biology from Tufts University in 1967 and her Ph.D. in Genetics from Cornell University Medical School in 1973. After completing her doctorate, she joined the Wadsworth Center, New York State Department of Health as a research scientist and worked there until illness forced her to retire in January 2006. Throughout her career, Lori focused on the mouse as a model system for human disease and played a key role in the continued success of mouse genetics research at Wadsworth Center. In addition to her NIH-funded research programs, Lori served in a number of administrative positions during the course of over 30 years at Wadsworth Center, including Chief of the Laboratory of Mammalian Genomics, Director of the Histocompatibility and Paternity Testing Laboratory, Director of the Molecular Genetics Program, Director of the Kidney Disease Institute and Chief of the Laboratory of Immunology. Most recently, she was instrumental in developing the Genomics Institute at Wadsworth Center and served as its Director from 1999 until her retirement. In addition to her Wadsworth-related duties, she was Professor of Biomedical Sciences at the University at Albany; she also held adjunct faculty positions at Albany Medical College and Rensselaer Polytechnic Institute, mentoring over two dozen postdoctoral fellows and graduate students during her career. She published over 140 scientific papers, held editorial posts with several peer-reviewed journals, served on advisory boards of a number of scientific organizations (e.g., Center for Rodent Genetics at NIEHS, Mouse Genome Informatics at The Jackson Laboratory) and served on NIH study sections for most of her career. Lori was well known for both her inquisitive mind and her love for cutting-edge technology. She continually searched for high-tech “toys” and made every attempt to bring all the latest technology in her field to the Wadsworth Center. She could easily be described as having multiple research careers. She first worked in immunogenetics, with particular emphasis on the major histocompatability complex, maintaining NIH grants for this research for over 20 years. In the next phase of her career, she examined the role of genetics, through mutagenesis studies, in other biological processes (e.g., development, differentiation). Finally, 10 years ago, she began to apply her expertise to the genetic dissection of mouse behavior. It is during this latter phase of her career that most IBANGS members came to know her. Active in our society for a number of years, she served as Secretary from 2002 to 2005 and recently received our first Lifetime Achievement Award (2005). She was also a regular participant in the Behavior Genetics Summer Schools and always enjoyed those sessions. She was never happier than when poring over data and discussing sometimes heatedly the results of a particular experiment. Even in the last few years, as her health deteriorated, her love of science and research kept her working at full capacity. Lori was sometimes “bigger than life” for those of us who knew her well, and her infectious laugh and scientific curiosity will be sorely missed.

Making and breaking serotonin neurons and autism

Dysfunction of brain serotonin system development is hypothesized to contribute to autistic behaviors. The testing of this hypothesis will likely depend on a better understanding of the genes and mechanisms involved in serotonin neuron cell fate specification. In this review we summarize the main features of vertebrate serotonin neuroanatomical development and recent studies that have revealed critical steps in the molecular genetic program that controls serotonin neuron phenotype. We then discuss the potential relevance of these findings to advances in autism research and to new molecular genetic tools under development that will impact future testing of the hypothesis.

EVALUATION OF OLIVE GERMPLASM IN IRAN ON THE BASIS OF MORPHOLOGICAL TRAITS: ASSESSMENT OF 'ZARD' AND 'ROWGHANI' CULTIVARS

EVALUATION OF OLIVE GERMPLASM IN IRAN ON THE BASIS OF MORPHOLOGICAL TRAITS: ASSESSMENT OF 'ZARD' AND 'ROWGHANI' CULTIVARS

Isolation of a deet-insensitive mutant of Drosophila melanogaster (Diptera: Drosophilidae).

Despite the widespread use of N,N,-diethyl-3-methylbenzamide (deet) in insect repellent products, nothing is known about the molecular basis for the repellency of deet, we initiated a molecular genetics program to elucidate the molecular mechanism of deet repellency in Drosophila melanogaster (Meigen). Deet repellency was apparently due to airborne vapors, as wild type flies were repelled by a deet-treated surface in the absence of physical contact and in the dark. A mutant was isolated using chemical mutagenesis and at choice assay. In a choice assay, mutant flies entered 82 +/- 1% of deet-containing tubes, whereas wild type flies entered only 6 +/- 2% of deet-containing tubes. The mutant was repelled by other repellents, benzaldehyde and citronellal. The mutation was recessive and located on the X chromosome.

Molecular genetic diagnostic program of multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma syndromes in Hungary.

Medullary thyroid carcinoma (MTC) occurs usually in sporadic form, but about a quarter of the cases are hereditary and appear as part of one of the multiple endocrine neoplasia type 2 (MEN2) syndromes. Mutations in the RET protooncogene are known to be the cause of the MEN2A and familial medullary thyroid carcinoma (FMTC) syndromes in the majority of the families. Direct DNA testing allows prophylactic thyroidectomy to be offered to individuals carrying a mutation in the above codons, and in mutation-negative cases it reduces the yearly screening-related burden on family members at risk of the disease. By DNA sequencing and PCR-restriction fragment length polymorphisms, 65 MTC probands were examined for mutations in residues 609, 611, 618, 620 of exon 10, and in residues 634, 768, 804 of exons 11, 13, and 14 respectively of the RET protooncogene. In our study, mutations in the above codons were detected in all of the 14 clinically MEN2A and FMTC families. One of these mutations, TGC609 TCC has not been reported previously. Of the 14 probands with the mutation, 25 relatives also had the identified mutation and 18 relatives proved to be non-carriers. Among the 51 probands with clinically sporadic MTC, none was found to carry a mutation in the above positions even if indirect signs of MTC, pheochromocytoma or hyperparathyroidism could be detected in some families. The frequency of the TGC634AGC mutation is unexpectedly high in our samples, which can probably be attributed to a founder effect. We conclude that screening for mutations in these codons is effective in families fulfilling the strict clinical criteria of MEN2A or FMTC.

Volvox as a Model System for Studying the Ontogeny and Phylogeny of Multicellularity and Cellular Differentiation

Volvox carteri, a spherical alga with a complete division of labor between approximately 2000 biflagellate somatic cells and 16 asexual reproductive cells called gonidia, provides a very attractive system for analyzing how a molecular-genetic program for cell-autonomous cellular differentiation may be encoded within a genome. Then, when considered in combination with a group of closely related volvocine algae that includes unicellular Chlamydomonas plus a series of colonial forms of increasing cell number and complexity, it also provides an attractive model system for analyzing how such a program for multicellularity and cytodifferentiation may have evolved. It is proposed that the following were some of the key steps in this evolutionary pathway: (1) The Chlamydomonas cell wall was transformed into an extracellular matrix (ECM) that joined sister cells into a colonial unit. (2) Larger organisms with more abundant ECM were favored because of the role the ECM plays in storing limiting nutrients. (3) In the V. carteri lineage the ancestral biphasic first biflagellate and then reproductive pathway of development became converted to a dichotomous pathway by introduction of two kinds of cell-type-specific negative regulators: one that blocked growth and reproduction in presumptive somatic cells and one that blocked somatic development in presumptive gonidia. Progress has been made in cloning and characterizing genes that are involved in setting apart the two cell lineages of V. carteri and in subsequently controlling their dichotomous differentiation. The strengths and weaknesses of V. carteri and its relatives as a model system for studying the evolution of multicellularity are discussed.

Cystic fibrosis: molecular diagnosis, population screening, and public policy.

○ Objective.-To review the current status of scientific knowledge and opinion regarding molecular genetic testing of mutations in the CFTR gene for purposes of diagnosis and population carrier screening of cystic fibrosis (CF). Data Sources.-Published research findings on the nature of the CFTR gene, pilot population screening studies in the United States and Europe, and ongoing deliberations of professional and governmental agencies considering implementation of widespread testing. Study Selection.-Findings relevant to the molecular heterogeneity of CFTR mutations and its implications for population carrier screening were considered. Data Extraction.-Information was extracted from studies published by us and others, as made available to recent consensus panels and professional committees. Data Synthesis.-These data were reevaluated in light of recent movements in professional and public policy regarding acceptability and desirability of widespread CF mutation testing. Effects to date of such testing on patient outcomes is reported. Conclusions.-The ability to test for CFTR mutations at the molecular level has already improved the diagnosis of symptomatic patients and expanded the reproductive options of family members of CF patients. The same technology also holds promise of identifying asymptomatic carriers and at-risk couples without family history in the general population so that they too might be offered prenatal diagnosis or other options. However, a number of key questions remain to be worked out before a widespread national screening program can be put into practice. These include the target population to be offered testing (the entire population vs high-risk ethnic groups), the size and nature of the mutation test panel (universal vs ethnic specific), the inclusion or exclusion of CFTR variants that do not cause classical CF, the optimal testing technology, appropriate standards for laboratory quality assurance, and the development of sufficient educational materials and genetic counseling resources for test delivery, reporting, and interpretation. The answers to these questions will be relevant not only to CF testing but also to many other large-scale molecular genetic screening programs being considered in the future.