Gene expression

The purpose of this study was to explore differences in perceived stress, glucocorticoid receptor (GR), and the expression of histone acetylation (HAT) of the corticotropin-releasing hormone (CRH) gene between non-Hispanic Black and non-Hispanic White women in their 2nd trimester of pregnancy. Black women are 2–3 times more likely to experience preterm birth (PTB) and maternal mortality than White women (Hoyert, 2022; Martin et al., 2019). Researchers have reported chronic stress associated with factors such as experiencing discrimination, financial hardship, and abuse may induce dysregulation of the stress hormones (Kramer et al., 2013; Shapiro-Mendoza et al., 2016). Likewise, the stress hormones glucocorticoid and CRH dysregulation have been linked to early labor, preeclampsia, and maternal death (Kramer et al., 2013; Shapiro-Mendoza et al., 2016; Yu et al., 2013). Additionally, social status, gender, education, and income are recognized as social determinants of health.
This study used an observational, cross-sectional design to analyze the differences in perceived stress, depression, GR, and HAT of the CRH gene between a group of Black and White pregnant women in their 2nd trimester of pregnancy. This study analyzed perceived stress, depression, and peripheral blood monocytes cells (PMBC) using secondary, deidentified data from pregnant women. The Perceived Stress Scale was used to measure stress, the profile in Mood Depression Scale measured depression, a chromatin immunoprecipitation (ChIP) to quantitative Polymerase chain reaction (qPCR) analysis was used to measure GR and HAT of the CRH gene. The data were analyzed using correlation and analysis of covariance (ANCOVA) to examine relationships and the differences between groups.

Clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas) protein system, CRISPR/Cas9, uses single-guide RNA to guide Cas9 to the target site for genome editing. In this study, the CRISPR/Cas9 system was used to knockout KED in tomato (Solanum lycopersicum). KED was first identified while screening the wounded tobacco (Nicotiana tabacum) leaves. We found that alignment of the protein sequence of SlKED (Solanum lycopersicum KED) and NtKED (Nicotiana tabacum KED) showed 55.1% identity. To investigate, we generated SlKED knockout tomato plants with a single base pair deletion, a five base pair deletion and a three base pair deletion with a single base pair insertion. We performed wounding assays and analyzed gene expression and found that the wounded SlKED knockout plant showed no gene induction. Furthermore, the biological assay results revealed that the tobacco hornworm (Manduca sexta) gained more mass when fed on the SlKED knockout plant. Our studies show that the KED gene plays a role in wound-induced mechanism and suggested it may involve in the plant defense system against biological stress and insect feeding.

Many different ways to create mutants have been established. This research
demonstrates yet another variation of the promoter tagging technique that allows for a
single step selection of the putative transgenic plants that have a mutation in
constitutively expressed genes. While tomato transformants have not yet been
convincingly confirmed, tobacco transformation resulted in seven transgenic lines
showing resistance to high concentrations of kanamycin. Two transgenic lines were
further investigated and three putative promoters isolated. Transient expression analysis
of leaves transformed by particle bombardment with vectors carrying beta-glucuronidase
gene driven by these putative promoters suggests two of them to be functional. Further
investigation is needed to confirm the expression in the stably transformed plants as well
as cloning of the genes downstream of the functional promoters and research of their
functions.

The Bel family of genes are fundamental to the apoptotic mechanism. Bcl-x a
member of this family, is alternatively spliced to create two main isoforms a long
(Bcl-xL) and a short (Bcl-xS) variant. The long form exhibits anti-apoptotic activity,
while the short form favors apoptosis. The proper balance of expression of these two
isoforms is crucial for several developmental processes such as thymic selection and
neural reshaping. A number of cancer types have been shown to over-express the long
form, thereby granting them some protection from apoptosis. To study the
transcriptional and post-transcriptional mechanisms regulating gene expression, the
Bcl-x gene has been utilized. A complex mini-gene construct has been create in order
to monitor the effects that promoter sequences, 5'UTR and 3'UTR's have on mRNA
splicing, RNA export, stability and translation. Abundant evidence exists indicating
that RNA processing events such as transcription, splicing and export are coupled, yet
the mechanisms and factors involved in regulating these processes are poorly
understood. The mini-gene is identical to the endogenous gene with the exception of a deletion to the 50Kb intron and the addition of a tag to differentiate the mini-gene
product from the endogenous mRNA and protein. This novel system allows for the
study of transcriptional and post-transcriptional mechanisms regulating gene
expression from RNA biogenesis on to the protein level.

In recent years more and more researchers have begun to use data mining and
machine learning tools to analyze gene microarray data. In this thesis we have collected a
selection of datasets revolving around prediction of patient response in the specific area
of breast cancer treatment. The datasets collected in this paper are all obtained from gene
chips, which have become the industry standard in measurement of gene expression. In
this thesis we will discuss the methods and procedures used in the studies to analyze the
datasets and their effects on treatment prediction with a particular interest in the selection
of genes for predicting patient response. We will also analyze the datasets on our own in
a uniform manner to determine the validity of these datasets in terms of learning potential
and provide strategies for future work which explore how to best identify gene signatures.

The ubiquitin ligase Highwire is responsible for cell-autonomously promoting
synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects
in synaptic function and extra branching at the axon terminal, corresponding to transient
branching that occur in the course of giant synapse formation during metamorphosis. The
MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor
Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner,
suggesting different molecular mechanisms downstream of the MAP kinase pathway
govern function and pruning. A novel role for Highwire is revealed, regulating the giant
fiber axon’s ability to respond to external cues regulated by Fos. When expression of the
transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline
glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous
manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology.

A naturally-occurring recessive lethal mutation in axolotls, Ambystoma mexicanum, is an intriguing model for studying tropomyosin expression regulation. Homozygous embryos(c/c) form hearts that are deficient in tropomyosin, lack organized myofibrils and fail to beat. Previous studies have shown that a non-coding RNA gene, MIR (Myofibril Inducing RNA), is sufficient to rescue the non-beating homozygous recessive mutant hearts by promoting sarcomeric tropomyosin expression that leads to formation of organized myofibrils and beating hearts. Real time RT-PCR reveals that mutant hearts express the same level mRNA of the alpha-tropomyosin and TM4 type tropomyosin (ATmC-3) gene as normal embryonic hearts. These genes show no differences with regard to the splicing patterns of normal and mutant. Using protease inhibitor LLnL and E-64d treatments and two-dimensional Western blots of normal and mutant hearts, it is found that mutant hearts express all tropomyosin protein isoforms as normal hearts but protein expression are at low levels. These studies suggest that there is a failure in the translational or posttranslational control mechanisms for tropomyosin protein synthesis in cardiac mutant axolotl hearts during development.

A recessive mutant gene, termed "c", for cardiac non-function in the Mexican axolotl, Ambystoma mexicanum, is responsible for the failure of myofibrillogenesis in cardiac nonfunction mutant embryonic hearts. Animals that are homozygous for the mutation (c/c) fail to develop beating hearts and consequently die. Thus, the Mexican axolotl has been a useful animal model to study embryonic heart development. Recently, the cardiac troponin T (cTnT) gene, along with three additional shorter isoforms of the gene, were cloned from normal embryonic hearts. These isoforms are believed to be the alternately spliced forms of the full length gene. One of the isoforms cloned is missing a cardiac-specific exon. Real-time PCR reveals that homozygous recessive mutant embryos (c/c) exhibit a lower transcription level of the cTnT gene than wildtype animals (+/+ or +/c). Expression levels of each of the isoforms are compared in normal and mutant hearts using quantitative real-time PCR.

Exoribonucleases degrade RNA and are important in RNA metabolism and gene expression. Mycoplasma genitalium, a bacterium with the smallest genome known, has only one identified exoribonuclease, RNase R (MgR). In this work RNA degradation properties of purified MgR were examined. As observed in Escherichia coli RNase R (EcR) studies, MgR degrades poly(A), rRNA, and oligoribonucleotides in 3'--->5' direction, though its substrate specificity and optimal activity requirements vary. Interestingly, MgR is sensitive to 2-O-methylation stopping downstream of such modifications in native rRNA and synthetic oligoribonucleotides. MgR removes the 3' trailer sequence from a tRNA precursor of M. genitalium and generates products equal to the mature tRNA, demonstrating a role of MgR in tRNA maturation. The 3' terminal CCA sequence and the acceptor stem of tRNA play a role in determining the formation of such products by MgR. These results suggest multiple functions of RNase R in RNA metabolism in Mycoplasma.