Characterization of CONSTANS, an Arabidopsis gene that promotes flowering.

Under long day conditions Arabidopsis thaliana plants grow vegetatively for approximately two weeks before they initiate flowering while under short day conditions the vegetative period lasts around six weeks. The constans (co) mutant flowers later than wild type under long days but at approximately the same time as wild type under short days. This suggests that the CO product promotes flowering in response to long days. The CO gene was cloned previously by chromosome walking and encodes a 373 amino acid protein containing two zinc fingers suggesting that it binds DNA and is involved in regulation of gene expression. A series of mutant alleles of the CO gene was characterized and the effect of each mutation on flowering time examined. All seven co mutants are semi-dominant, with the heterozygotes having flowering times and leaf numbers intermediate between wild type and the homozygous co parent, and all differ quantitatively with respect to flowering time and leaf number. The co alleles were characterized at the molecular level and the mutations were clustered into two regions of the gene that define important domains of the predicted CO protein. Five of the mutations affect the zinc finger region near the N-terminus of the protein and the remaining two mutations affect adjacent amino acids in a basic domain near the C-terminus of the protein. Both of these domains and the individual amino acids affected by the mutations are highly conserved. Homology searches revealed a family of at least 14 closely related genes in Arabidopsis and homologues in a number of other plant species. The seven co mutations all cause in-frame changes and none were unambiguous null alleles. To investigate the effect of loss of CO function a putative null allele, co-8, was isolated after gamma-irradiation. This was characterized at the molecular level and found to contain a 1.3 kb deletion which removes approximately 1 kb of the promoter and 0.3 kb of the coding sequence at the 5’ end of the CO ORF that includes the zinc finger domain. The new co-8 mutant has an intermediate flowering time with respect to the seven previously isolated co mutants, and is also semi-dominant. A number of constructs designed to express derivatives of the CO protein were introduced into Arabidopsis to investigate whether it was possible to repress flowering by the creation of dominant negative mutations. However, with the exception of one or two small effects these did not greatly affect flowering time. A Brassica napus homologue of the CO gene was isolated. The predicted protein is 74% identical to the Arabidopsis CO protein, with the greatest conservation over the zinc fingers (91% identical) and C-terminal basic domain (94% identical). The homologue was able to complement the Arabidopsis co-2 mutation and restore wild type flowering time

Mining genome data for endogenous viral elements and interferon stimulated genes: insights into host virus co-evolution

Paleovirology is the study of viruses over evolutionary timescales. Contemporary paleovirological analyses often rely on sequence data, derived from organism genome assemblies. These sequences are the germline inherited remnants of past viral infection, in the form of endogenous viral elements and the host immune genes that are evolving to combat viruses. Their study has found that viruses have exerted profound influences on host evolution, and highlighted the conflicts between viruses and host immunity. As genome sequencing technology cheapens, the accumulation of genome data increases, furthering the potential for paleovirological insights. However, data on ERVs, EVEs and antiviral gene evolution, are often not captured by automated annotation pipelines. As such, there is scope for investigations and tools that investigate the burgeoning bulk of genome data for virus and and antiviral gene sequence data in the search of paleovirological insight

POST-TRANSCRIPTIONAL REGULATION OF AFP AND IgM GENES

Gene expression can be regulated at multiple steps once transcription is initiated. I have studied two different gene models, the α-Fetoprotein (AFP) and the immunoglobulin heavy chain (IgM) genes, to better understand post-transcriptional gene regulation mechanisms. The AFP gene is highly expressed during fetal liver development and dramatically repressed after birth. There is a mouse strain-specific difference between adult levels of AFP, with BALB/cJ mice expressing 10 to 20-fold higher levels compared to other mouse strains. BALB/cJ mice express low levels of Zhx2 and thus incompletely repress AFP. Despite differences in steady state AFP mRNA levels in the adult liver between Balb/cJ and wild-type mice, transcription rates across this gene were similar, indicating a post-transcriptional regulatory mechanism. I found accumulated unspliced RNA across multiple AFP introns in wild-type mice where mature AFP mRNA levels are low, suggesting overall AFP splicing is inefficient in the presence of Zhx2. The IgM gene is alternative processed to produce two mRNA isoforms through a competition between cleavage/polyadenylation (μspA) and splicing reactions and the pA/splice RNA expression ratio increases during B cell maturation. Cotranscriptional cleavage (CoTC) events, driven by specific cis-acting elements, are required downstream of some poly(A) signals to terminate transcription. In some cases, a pause site can produce similar effect. I explored whether there is a CoTC-like element within the IgM gene that may contribute to developmental changes in the mRNA ratio. In both a B cell and plasma cell line there was a gradual decrease in transcripts downstream from the μspA signal, suggesting that there is not evidence for a CoTC element within the IgM gene. To examine the effect a CoTC element would have on the competition between the splice and μspA reactions, we inserted the CoTC sequence of the β-globin gene into different locations downstream of the μspA signal. While the β-globin CoTC element caused cotranscriptional cleavage in all locations, it only affected the μspA/splice ratio when located close to the μspA site. This suggests there is a position effect of the inserted CoTC element on the competing polyadenylation and splicing reactions within the IgM transcripts

Abstracts from the 19th FGC

Plenary and poster session abstracts from the 19th Fungal Genetics Conferenc

Analysis of Expressed Sequence Tags Mapping to the Critical Region of the 5q- Syndrome

The 5q- syndrome is a myelodysplastic syndrome characterised by a macrocytic anaemia, hypolobulated megakaryocytes, a low risk of transformation to AML, and a 5q- chromosome as the sole karyotypic abnormality. The approximate 5Mb critical region of gene loss of the 5q- syndrome has been defined in two patients with the 5q- syndrome at 5q31-q33, flanked by the genes for FGF1 and IL12ß. The frequent loss of genetic material from the long arm of chromosome 5 in association with a malignancy has led to the hypothesis that, by analogy with other malignancies characterised by genetic loss, the 5q- syndrome is caused by loss of function of a gene with tumour suppressor activity. A transcript map of the 5q- syndrome critical region was generated with the aim of identifying the putative tumour suppressor gene associated with this disease. The expressed sequence tag (EST) database, db(EST) was used to isolate novel coding sequences mapping to the critical region of gene loss. Ten novel coding sequences (C5orf4, AF010242, AF156165, Cdy-17a06, Bda-87b11 195312, 4885953 / 143772,120101,195971, and 199067) were localised to the YAC contig spanning the critical region at 5g31-q33. The ten cDNA clones were sequenced, and overlapping clones were identified and sequenced in order to generate complete or partial coding sequences. This included the cloning of novel gene, C5orf4, and the identification of the human synaptopodin and dynactin p62 genes. In addition, the human homologues of the Drosophila melanogaster RMSA-1 and Saccharomyces cerevisiae CDC60 genes, and two known human genes (PP2A and HAH1) were localised to the critical region. Expression in human peripheral blood leukocytes and CD34+ progenitor cells was investigated for each known and novel gene. Genomic localisation, expression patterns and predicted function would suggest these known and novel genes represent putative tumour suppressor genes. Mutation studies were carried out on six known, and two novel candidate genes mapping to the narrowed 1.5Mb critical region of gene loss at 5g31.3-q32. No mutations were found in the coding regions/exons of these genes, suggesting they are not involved in the pathogenesis of the 5q- syndrome

The molecular basis of childhood nephrotic syndrome.

Childhood nephrotic syndrome results from massive leakage of protein into the urine, a low plasma albumin and oedema. Disease may be kidney-specific, occur as part of a malformation syndrome, or may complicate systemic diseases such as diabetes mellitus. Despite the apparent heterogeneity, the underlying defect is loss of the normal permselective characteristics of the glomerular filtration barrier (GFB). Clues for a molecular basis came from observation of occasional autosomal dominant or recessive inheritance, and the detection of WT1 mutations in Denys Drash syndrome (DDS), a triad of intersex, nephrotic syndrome and Wilms' tumour (Pelletier et al, 1991). The role of three glomerular genes WTl, NPHS1 and NPHS2 in the pathogenesis of glomerular protein leak was investigated. WTl mutations were not detected in non- syndromic diffuse mesangial sclerosis (DMS) and focal segmental glomerulosclerosis (FSGS), despite their association with DDS. However, subsequent analysis established that WTl mutations cause Frasier syndrome, a triad of FSGS, intersex and gonadoblastoma, by reversing the normal +(KTS)/-(KTS) WTl isoform ratio. Unfortunately, yeast 2-hybrid screens failed to ascertain any WTl protein binding partners with clear roles in glomerular function, and through which the effects of mutations might be mediated. A wide range of NPHS1 mutations was detected in Finnish type congenital nephrotic syndrome (CNF) in non-Finns, and a novel mild CNF phenotype described. NPHS2 mutations affected some CNF cases, and an overlap in the NPHS1/NPHS2 mutation spectrum was confirmed by the discovery of a unique di-genic inheritance of mutations. This modified the phenotype from CNF to congenital FSGS, providing the first evidence for a functional inter-relationship between these genes. Finally, disrupted protein-DNA binding to an area of the NPHS1 promoter containing a G->C base substitution was identified, suggesting the location of a transcription factor binding site and underscoring the importance of appropriate transcriptional control of NPHS1 for correct gene function

Genetic and environmental control of lignin biosynthesis and C emission from crop stover

Lignin is the second most abundant terrestrial biopolymer in the world and provides structural strength to plants. The maize brown midrib (bm) mutants accumulate less and altered lignin relative to non-mutants. Maize bm4-encoded folylpolyglutamate synthase functions to generate the preferred substrate of folate-dependent enzymes, such as bm2-encoded methylenetetrahydrofolate reductase. Consistent with bm4’s predicted function upstream of bm2, both mutants display a 7-10% reduction in lignin content and a 16-40% increase in S/G lignin ratio relative to wild-type. This interplay between lignin biosynthesis and various metabolic processes is also seen between lignin and stover carbon emission. As climate change intensifies, the continued accumulation of CO2 in the atmosphere poses potentially irreversible threats to the environment and economy. Exploiting the natural carbon cycle of plants to sequester excess atmospheric C in the soil is a promising strategy for climate remediation. To assess the potential of this approach, stover from over 6,000 maize and sorghum genotypes was incubated in soil and assayed for C emission. While compositional traits, such as lignin, explain up to 48% of variation in C emission, environment still plays a major role. Dynamics of C emission vary across environments and seem to be, at least partially, controlled by the age and lignin content of the stover assayed. Despite environmental influence on C emission, adequate genetic control exists for this trait to allow for moderate heritability estimates and genomic prediction accuracy. Additionally, GWAS for C emission identifies numerous promising candidate genes that could serve as breeding targets for generating varieties with enhanced carbon sequestration potential

Analysis of transcription factor CREM dependent gene expression during mouse spermatogenesis

Computational methods are getting increasingly important for the analysis of large data sets in molecular biology. The data sets analyzed in this thesis are derived from experiments measuring the changes of expression levels in response to the transcription factor CREM (cAMP Responsive Element Modulator) during mouse spermatogenesis. In the course of this analysis new computational methods were developed and used that will also be of value in other projects in Bioinformatics. CREM belongs to a family of cAMP-responsive nuclear factors. The activator splice-isoform CREM is exclusively expressed at high levels in post-meiotic germ cells during mouse spermiogenesis. Mutant male mice lacking CREM expression are sterile due to lack of maturation of the germ cells. In order to find CREM target genes the mRNA expression levels in testes of CREM-deficient mice and wild-type mice were compared using the suppression subtractive hybridization (SSH) technique as well as oligonucleotide DNA microarrays. SSH was used to selectively amplify the differentially expressed genes. 12,000 clones, which contain sequence fragments of genes expressed stronger in wild-type as in the CREM (-/-) mutant, were analyzed by a combination of sequencing and hybridization. Sequence analysis methods were used to characterize 956 unique sequences. Homologies to 158 known mouse genes and 99 known genes from other organisms were detected. 296 sequences show homologies to sequences of expressed sequence tags (ESTs). 199 novel sequences have been found. The sequences not corresponding to full length genes of known function were characterized using publicly available EST data. To make EST databases useful for data analysis all of the publicly available ESTs have been grouped into clusters and methods to analyze and visualize EST data were developed. Nylon cDNA microarrays containing the unique sequences from the CREM SSH library were constructed to determine expression levels of those sequences. Most of the sequences from the CREM SSH library are shown to be expressed in wild-type but are down-regulated in CREM deficient mice. Statistical methods to standardize microarray expression data were developed and software was implemented to perform comparisons. Further CREM dependent genes were detected comparing the mRNA expression levels in testes of CREM deficient mice and wild-type mice using Affymetrix oligonucleotide microarrays containing 10,000 mouse sequences. Comparison of the different techniques (SSH, nylon cDNA arrays and Affymetrix oligonucleotide microarrays) shows that the results are complementing each other. The unique sequences from the CREM SSH library were further analyzed by determining the spermatogenic stage specific expression profiles. cDNA from prepubertal mice at certain stages of spermatogenesis were hybridized on nylon cDNA arrays. Several important functional groups of genes like transcription factors, signal transduction proteins and metabolic enzymes are shown to be coexpressed at the latest stages of spermatogenesis. Expression profiles were arranged to find similar profile shapes and co-regulation of functionally related genes. An algorithm to arrange the profiles in an optimal linear order was developed. The linear order is constructed in a way that similar expression profiles end up close together in the linear order, i.e. the sum over all distances of neighboring profiles is minimized. This corresponds to the solution of a traveling salesman problem (TSP), which is well known in computer science. A fast algorithm that computes a heuristic solution to a TSP was adapted to be used in expression profile analysis

XXIII Fungal Genetics Conference

Program and abstracts from the 23rd Fungal Genetics Conference and Poster Abstracts at Asilomar, March 15-20, 200