The document summarizes research into the roles of genes ELL2 and ELL3 in plasma cell development. It describes how ELL2 expression increases and ELL3 decreases as B cells differentiate into antibody-secreting plasma cells. The research aimed to determine if ELL1 and ELL3 have similar functions to ELL2. Cell lines expressing only ELL2 or ELL3 were analyzed to confirm expression. A lentivirus was prepared to introduce genes into B cells from ELL2 knockout mice to test for functional complementation. The work established methods and preliminary results to investigate the roles of the ELL genes in plasma cell development.
This document summarizes a study investigating the oncoprotein EVI1 and its regulation of the carbonic anhydrase 3 (CA3) gene. Microarray analysis identified CA3 as a major target of EVI1. The study aims to validate changes in CA3 expression and determine the mechanism and significance of EVI1-mediated transcriptional regulation of CA3. Results show that EVI1 suppresses CA3 expression at both the mRNA and protein levels by decreasing CA3 promoter activity. This suppression of the antioxidant CA3 gene leads to increased caspase activity and apoptosis in cancer cells expressing EVI1.
1. The document discusses the multigene organization of immunoglobulins, including the lambda and kappa light chains and heavy chains.
2. It describes how each contains multiple gene segments (V, D, J, C) that rearrange during B cell maturation to generate the variable and constant regions that determine antibody specificity.
3. The gene segments are located on different chromosomes and rearrangement of V, D, J segments in heavy chains and V, J segments in light chains creates enormous antibody diversity.
1) The study found that IKKα, like IKKβ and NEMO/IKKγ, is required for the activation of NF-κB target genes in response to TNFα and IL-1 stimulation in mouse embryonic fibroblasts.
2) DNA microarray analysis identified many known and novel NF-κB dependent target genes that were regulated by all three subunits of the IKK complex.
3) Some NF-κB target genes were dependent on the IKKs even in the absence of extracellular stimuli, suggesting the IKK complex also regulates basal levels of NF-κB activity.
This document discusses oxidoreductases, enzymes that catalyze the formation of disulfide bonds, in Neisseria meningitidis. N. meningitidis contains three oxidoreductases, NmDsbA1, NmDsbA2, and NmDsbA3, which share varying levels of amino acid homology. While NmDsbA1 and NmDsbA3 can catalyze disulfide bonds in some substrates like PilE and PilQ, NmDsbA3 cannot catalyze bonds in these substrates. The document examines the structures and functions of the three oxidoreductases to better understand their differing substrate specificities.
This document discusses the role of somatic mutagenesis in the development of autoimmunity, specifically in systemic lupus erythematosus (SLE). It argues that:
1) Antinuclear antibodies (ANA) that arise in SLE predominantly originate from non-autoreactive B cells that undergo somatic hypermutation (SHM) and are transformed into autoreactive cells, rather than arising from autoreactive B cells escaping central tolerance.
2) SHM may create antigenic peptides in the B cell receptor that provide a route for unregulated T cell help to autoreactive B cells.
3) Spontaneous somatic mutagenesis of genes regulating B cell survival and activation may be a stochastic rate-
1. The document summarizes research on CLN7 disease, which is caused by mutations in the CLN7 gene encoding a lysosomal membrane protein.
2. A mouse model of CLN7 disease was generated that recapitulates some neuropathological features seen in human patients, including accumulation of autofluorescent storage material in the brain.
3. The researchers aim to identify the pathomechanisms of CLN7 disease, develop biomarkers, analyze the function of the CLN7 protein, and understand how mutations impact its localization and function to inform potential therapies.
This document discusses several topics related to genes and genomes. It begins by explaining that one gene codes for one enzyme or polypeptide. It then discusses genetic dominance being explained by mutant protein properties. Later sections discuss gene expression requiring several processes, and that some DNA sequences can code for more than one polypeptide. The document also examines that eukaryotic genomes contain both repetitive and nonrepetitive DNA sequences.
This document summarizes a study investigating the oncoprotein EVI1 and its regulation of the carbonic anhydrase 3 (CA3) gene. Microarray analysis identified CA3 as a major target of EVI1. The study aims to validate changes in CA3 expression and determine the mechanism and significance of EVI1-mediated transcriptional regulation of CA3. Results show that EVI1 suppresses CA3 expression at both the mRNA and protein levels by decreasing CA3 promoter activity. This suppression of the antioxidant CA3 gene leads to increased caspase activity and apoptosis in cancer cells expressing EVI1.
1. The document discusses the multigene organization of immunoglobulins, including the lambda and kappa light chains and heavy chains.
2. It describes how each contains multiple gene segments (V, D, J, C) that rearrange during B cell maturation to generate the variable and constant regions that determine antibody specificity.
3. The gene segments are located on different chromosomes and rearrangement of V, D, J segments in heavy chains and V, J segments in light chains creates enormous antibody diversity.
1) The study found that IKKα, like IKKβ and NEMO/IKKγ, is required for the activation of NF-κB target genes in response to TNFα and IL-1 stimulation in mouse embryonic fibroblasts.
2) DNA microarray analysis identified many known and novel NF-κB dependent target genes that were regulated by all three subunits of the IKK complex.
3) Some NF-κB target genes were dependent on the IKKs even in the absence of extracellular stimuli, suggesting the IKK complex also regulates basal levels of NF-κB activity.
This document discusses oxidoreductases, enzymes that catalyze the formation of disulfide bonds, in Neisseria meningitidis. N. meningitidis contains three oxidoreductases, NmDsbA1, NmDsbA2, and NmDsbA3, which share varying levels of amino acid homology. While NmDsbA1 and NmDsbA3 can catalyze disulfide bonds in some substrates like PilE and PilQ, NmDsbA3 cannot catalyze bonds in these substrates. The document examines the structures and functions of the three oxidoreductases to better understand their differing substrate specificities.
This document discusses the role of somatic mutagenesis in the development of autoimmunity, specifically in systemic lupus erythematosus (SLE). It argues that:
1) Antinuclear antibodies (ANA) that arise in SLE predominantly originate from non-autoreactive B cells that undergo somatic hypermutation (SHM) and are transformed into autoreactive cells, rather than arising from autoreactive B cells escaping central tolerance.
2) SHM may create antigenic peptides in the B cell receptor that provide a route for unregulated T cell help to autoreactive B cells.
3) Spontaneous somatic mutagenesis of genes regulating B cell survival and activation may be a stochastic rate-
1. The document summarizes research on CLN7 disease, which is caused by mutations in the CLN7 gene encoding a lysosomal membrane protein.
2. A mouse model of CLN7 disease was generated that recapitulates some neuropathological features seen in human patients, including accumulation of autofluorescent storage material in the brain.
3. The researchers aim to identify the pathomechanisms of CLN7 disease, develop biomarkers, analyze the function of the CLN7 protein, and understand how mutations impact its localization and function to inform potential therapies.
This document discusses several topics related to genes and genomes. It begins by explaining that one gene codes for one enzyme or polypeptide. It then discusses genetic dominance being explained by mutant protein properties. Later sections discuss gene expression requiring several processes, and that some DNA sequences can code for more than one polypeptide. The document also examines that eukaryotic genomes contain both repetitive and nonrepetitive DNA sequences.
This document summarizes research on lysosomal membrane permeability (LMP) and its implications for neurodegenerative diseases like neuronal ceroid lipofuscinoses (NCLs). The researchers found evidence that LMP occurs in mouse models of two forms of NCL, CLN2 and CLN3, resulting in the formation of protein aggregates. Normally, LMP triggers a response to sequester damaged lysosomes, but this "lysophagy" response is impaired in NCL models. As a result, aggregates containing lysosomal contents accumulate and may contribute to neurodegeneration. The researchers are testing the drug arimoclomol, which could prevent LMP, in hopes of reducing neurodegeneration in CL
The document discusses the mechanisms by which the immune system generates antibody diversity to recognize millions of antigens. It describes 7 mechanisms: 1) use of multiple gene segments for light and heavy chains, 2) combinatorial V-J and V-D-J joining of gene segments, 3) addition of P-nucleotides and N-nucleotides during joining to introduce junctional diversity, 4) junctional flexibility in joining segments, 5) combinatorial association of paired heavy and light chains, 6) somatic hypermutation of variable regions within germinal centers, and 7) clonal selection of B cells with higher affinity antibodies. Together, these mechanisms allow the immune system to produce a vast repertoire of antibodies despite having relatively few antibody gene segments
This study investigated the role of the CLN5 and CLN8 proteins in sphingolipid metabolism and ceramide synthesis. The following key findings are reported:
1) CLN5-deficient fibroblasts showed decreased levels of ceramide, sphingomyelin, and glycosphingolipids as well as defects in adhesion, increased growth, and apoptosis.
2) Expression of CLN8 protein corrected the growth and apoptosis abnormalities in CLN5-deficient cells, suggesting CLN5 and CLN8 proteins interact and modulate ceramide synthase activity.
3) Both CLN5- and CLN8-deficient cells showed decreased de novo ceramide synthesis and specific ceramide
The document discusses the molecular basis of antibody diversity. It describes how antibodies are produced by plasma cells and recognize and bind antigens. The immunoglobulin gene families for the heavy and light chains are located on different chromosomes and contain multiple gene segments. Gene rearrangement at the DNA and RNA level brings together gene segments to generate a diverse repertoire of antibodies. Mechanisms like combinatorial joining of variable region genes, junctional flexibility, and addition of nucleotides further increase diversity, as does somatic hypermutation in response to antigens. The associations between rearranged heavy and light chains also contribute to diversity. This molecular basis has enabled applications like monoclonal antibodies.
Antibody class switching is a biological mechanism that changes a B cell's production of antibodies from one class to another, such as from IgM to IgG. It involves changing the constant region of the antibody heavy chain through DNA recombination, while retaining the same variable region and antigen specificity. Double-stranded breaks are generated in switch regions of DNA upstream of constant region genes. DNA is cleaved at two switch regions and the intervening DNA is deleted, allowing substitution of a different constant region exon and changing the class of antibody produced. The free DNA ends are joined by non-homologous end joining to link the variable region to the new constant region gene. This allows a B cell to produce different antibody classes while recognizing
- Antibodies are Y-shaped proteins produced by B cells that recognize and bind to pathogens. They have great diversity and specificity that allows them to target a wide range of antigens.
- Antibodies are composed of two heavy chains and two light chains that form an antigen binding site. The variable regions of the heavy and light chains are responsible for binding to different antigens.
- Monoclonal antibodies are produced from a single clone of B cells and have identical antigen specificity, allowing them to be used as standardized reagents for research and medical purposes. They are produced by fusing B cells with myeloma cells to form immortal hybridomas.
nucleic acid
history
central dogma of life
types of nucleic acid
functions of DNA
Replication
encoding information
mutation and recombination
gene expression
Development of an_immunogenicity_score_for_hla-dq_Georgia Marcusso
Eplets are defined as distinct amino acid configurations on the surface of HLA
molecules. The aim of this study was to estimate the immunogenicity of HLADQ eplets in a cohort of 221 pregnancies with HLA-DQ mismatches
This document summarizes research into the interaction between two proteins, SLP76 and ADAP, that are critical for T-cell signaling. The researcher generated fluorescent protein-tagged constructs of ADAP with mutations to phosphotyrosine residues that are hypothesized to bind SLP76. When these ADAP constructs were transfected into T-cells, immunoprecipitation showed the wild-type ADAP bound SLP76, but binding was too low to detect with mutants. Future work will optimize the assay and examine interactions between SLP76 and ADAP mutants to understand how multiple binding sites promote T-cell activation.
The researchers are studying synaptic changes in a fruit fly model of variant infantile Batten disease, which is caused by mutations in the CLN7 gene. They found that fruit flies lacking the CLN7 gene had smaller nerves and fewer synapses compared to normal flies. When the CLN7 gene was restored, the nerve returned to normal size. This demonstrates that losing the CLN7 gene causes the changes seen in nerve size and number of synapses. The researchers aim to understand what the CLN7 gene does in nerve cells and how its mutation leads to neurodegeneration, in order to inform future therapy development.
Generation of Antibody Diversity- Quick revision from Kuby through presentationSharmistaChaitali
Immunology, Kuby's fifth edition notes for strong background in the topic, General introduction, Types of Antibody and Structure, Experiments, Mechanisms
This document discusses various topics related to transcriptomics and lexico-syntactic analysis including:
- The challenges of resolving gene names and identifiers from multiple databases.
- Using text mining of biomedical abstracts to identify regulatory relationships between genes/proteins by parsing sentences and identifying relationships.
- Integrating gene expression data from multiple experiments and species to infer functional links between genes based on correlations in expression profiles.
- Other related resources discussed include the STRING database for predicting protein-protein interactions and the use of gene synonyms to integrate different types of biological data.
The genetic code is a dictionary that translates nucleotide sequences into amino acid sequences. It is composed of 64 codons that are read in groups of three nucleotides. The genetic code is universal, unambiguous, redundant, and non-overlapping. It specifies 20 standard amino acids through 61 codons, while 3 codons are stop signals that terminate protein synthesis. The code allows for wobbling in base pairing at the third position of codons, increasing decoding efficiency. Mutations can alter protein sequences through changes to codons.
1) Researchers created a cloning vector for the methanogen Methanosarcina acetivorans C2A to measure gene expression. The vector included the lacZ gene and native pmcrB promoter but was uncertain about the origin of replication.
2) Two cloning methods were attempted - Gibson assembly and PCR sewing with restriction enzymes. Gibson assembly failed to ligate correctly while PCR sewing excluded a small DNA segment but maintained the correct sequence otherwise.
3) Beta-galactosidase assays will be used to quantitatively measure protein expression of the introduced lacZ gene. Preliminary assays showed the method works for a shuttle plasmid and that the native methanogen has no background lacZ activity. The recombinant plasmid created
- ALK gene mutations can cause lymphomas such as ALCL and rare cases of DLBCL. The ALK gene encodes a tyrosine kinase receptor important for neural development. In ALCL, the ALK gene often fuses with other genes like NPM, forming chimeric proteins that drive uncontrolled cell growth. ALCL with ALK mutations has a better prognosis and responds well to chemotherapy, with many patients achieving long-term remission or cure. New targeted therapies that inhibit ALK are also under development.
Antibodies, also known as immunoglobulins, are glycoproteins produced by B cells that bind to specific antigens. There are five classes of immunoglobulins - IgG, IgM, IgD, IgA, and IgE - which differ in their heavy chains. Immunoglobulins have two primary functions: antigen binding and effector functions like complement fixation and binding to immune cells. Their structure consists of two light chains and two heavy chains that form a Y-shape, with constant and variable regions that determine the class and allow binding to different antigens.
The document discusses the generation of antibody diversity in the immune system. It explains that there are millions of possible antigens but only a small number of immunoglobulin genes in our genome. Through seven mechanisms, including multiple germline genes, combinatorial V(D)J joining, junctional flexibility, and somatic hypermutation, the immune system is able to generate a diverse repertoire of antibodies against all potential antigens from a limited set of gene segments. These mechanisms operate during B cell development and maturation in the bone marrow and lymphoid tissues.
The document summarizes the key mechanisms by which the human immune system generates a diverse repertoire of antibodies from a relatively small number of genes. It describes the somatic variation theory where mutation and recombination of immunoglobulin genes in somatic cells results in high antibody diversity. It explains processes like V(D)J recombination of light and heavy chain genes, junctional diversity, allelic exclusion, somatic hypermutation, and class switching which all contribute to antibody diversity.
The document provides an overview of apoptosis, or programmed cell death, discussing its molecular mechanisms and role in development and disease. It summarizes that apoptosis occurs through intrinsic and extrinsic pathways, is regulated by Bcl-2 family proteins like Bax and Bcl-2, and involves caspase activation leading to DNA fragmentation and phagocytosis of cell fragments. The document also discusses the importance of apoptosis in immune system development and its relevance to cancer.
(1) The study aimed to define the structure of the MLIP gene and its tissue-specific expression patterns.
(2) PCR and western blot experiments showed that MLIP exon 1a is expressed in heart and skeletal muscle, while exon 1b is expressed in the brain. Neither exon is expressed in the pancreas or liver.
(3) This suggests MLIP uses different promoters in different germ layers - exon 1a promoter in mesoderm, exon 1b promoter in ectoderm, and unknown promoters in endoderm tissues like pancreas and liver.
This document summarizes research on lysosomal membrane permeability (LMP) and its implications for neurodegenerative diseases like neuronal ceroid lipofuscinoses (NCLs). The researchers found evidence that LMP occurs in mouse models of two forms of NCL, CLN2 and CLN3, resulting in the formation of protein aggregates. Normally, LMP triggers a response to sequester damaged lysosomes, but this "lysophagy" response is impaired in NCL models. As a result, aggregates containing lysosomal contents accumulate and may contribute to neurodegeneration. The researchers are testing the drug arimoclomol, which could prevent LMP, in hopes of reducing neurodegeneration in CL
The document discusses the mechanisms by which the immune system generates antibody diversity to recognize millions of antigens. It describes 7 mechanisms: 1) use of multiple gene segments for light and heavy chains, 2) combinatorial V-J and V-D-J joining of gene segments, 3) addition of P-nucleotides and N-nucleotides during joining to introduce junctional diversity, 4) junctional flexibility in joining segments, 5) combinatorial association of paired heavy and light chains, 6) somatic hypermutation of variable regions within germinal centers, and 7) clonal selection of B cells with higher affinity antibodies. Together, these mechanisms allow the immune system to produce a vast repertoire of antibodies despite having relatively few antibody gene segments
This study investigated the role of the CLN5 and CLN8 proteins in sphingolipid metabolism and ceramide synthesis. The following key findings are reported:
1) CLN5-deficient fibroblasts showed decreased levels of ceramide, sphingomyelin, and glycosphingolipids as well as defects in adhesion, increased growth, and apoptosis.
2) Expression of CLN8 protein corrected the growth and apoptosis abnormalities in CLN5-deficient cells, suggesting CLN5 and CLN8 proteins interact and modulate ceramide synthase activity.
3) Both CLN5- and CLN8-deficient cells showed decreased de novo ceramide synthesis and specific ceramide
The document discusses the molecular basis of antibody diversity. It describes how antibodies are produced by plasma cells and recognize and bind antigens. The immunoglobulin gene families for the heavy and light chains are located on different chromosomes and contain multiple gene segments. Gene rearrangement at the DNA and RNA level brings together gene segments to generate a diverse repertoire of antibodies. Mechanisms like combinatorial joining of variable region genes, junctional flexibility, and addition of nucleotides further increase diversity, as does somatic hypermutation in response to antigens. The associations between rearranged heavy and light chains also contribute to diversity. This molecular basis has enabled applications like monoclonal antibodies.
Antibody class switching is a biological mechanism that changes a B cell's production of antibodies from one class to another, such as from IgM to IgG. It involves changing the constant region of the antibody heavy chain through DNA recombination, while retaining the same variable region and antigen specificity. Double-stranded breaks are generated in switch regions of DNA upstream of constant region genes. DNA is cleaved at two switch regions and the intervening DNA is deleted, allowing substitution of a different constant region exon and changing the class of antibody produced. The free DNA ends are joined by non-homologous end joining to link the variable region to the new constant region gene. This allows a B cell to produce different antibody classes while recognizing
- Antibodies are Y-shaped proteins produced by B cells that recognize and bind to pathogens. They have great diversity and specificity that allows them to target a wide range of antigens.
- Antibodies are composed of two heavy chains and two light chains that form an antigen binding site. The variable regions of the heavy and light chains are responsible for binding to different antigens.
- Monoclonal antibodies are produced from a single clone of B cells and have identical antigen specificity, allowing them to be used as standardized reagents for research and medical purposes. They are produced by fusing B cells with myeloma cells to form immortal hybridomas.
nucleic acid
history
central dogma of life
types of nucleic acid
functions of DNA
Replication
encoding information
mutation and recombination
gene expression
Development of an_immunogenicity_score_for_hla-dq_Georgia Marcusso
Eplets are defined as distinct amino acid configurations on the surface of HLA
molecules. The aim of this study was to estimate the immunogenicity of HLADQ eplets in a cohort of 221 pregnancies with HLA-DQ mismatches
This document summarizes research into the interaction between two proteins, SLP76 and ADAP, that are critical for T-cell signaling. The researcher generated fluorescent protein-tagged constructs of ADAP with mutations to phosphotyrosine residues that are hypothesized to bind SLP76. When these ADAP constructs were transfected into T-cells, immunoprecipitation showed the wild-type ADAP bound SLP76, but binding was too low to detect with mutants. Future work will optimize the assay and examine interactions between SLP76 and ADAP mutants to understand how multiple binding sites promote T-cell activation.
The researchers are studying synaptic changes in a fruit fly model of variant infantile Batten disease, which is caused by mutations in the CLN7 gene. They found that fruit flies lacking the CLN7 gene had smaller nerves and fewer synapses compared to normal flies. When the CLN7 gene was restored, the nerve returned to normal size. This demonstrates that losing the CLN7 gene causes the changes seen in nerve size and number of synapses. The researchers aim to understand what the CLN7 gene does in nerve cells and how its mutation leads to neurodegeneration, in order to inform future therapy development.
Generation of Antibody Diversity- Quick revision from Kuby through presentationSharmistaChaitali
Immunology, Kuby's fifth edition notes for strong background in the topic, General introduction, Types of Antibody and Structure, Experiments, Mechanisms
This document discusses various topics related to transcriptomics and lexico-syntactic analysis including:
- The challenges of resolving gene names and identifiers from multiple databases.
- Using text mining of biomedical abstracts to identify regulatory relationships between genes/proteins by parsing sentences and identifying relationships.
- Integrating gene expression data from multiple experiments and species to infer functional links between genes based on correlations in expression profiles.
- Other related resources discussed include the STRING database for predicting protein-protein interactions and the use of gene synonyms to integrate different types of biological data.
The genetic code is a dictionary that translates nucleotide sequences into amino acid sequences. It is composed of 64 codons that are read in groups of three nucleotides. The genetic code is universal, unambiguous, redundant, and non-overlapping. It specifies 20 standard amino acids through 61 codons, while 3 codons are stop signals that terminate protein synthesis. The code allows for wobbling in base pairing at the third position of codons, increasing decoding efficiency. Mutations can alter protein sequences through changes to codons.
1) Researchers created a cloning vector for the methanogen Methanosarcina acetivorans C2A to measure gene expression. The vector included the lacZ gene and native pmcrB promoter but was uncertain about the origin of replication.
2) Two cloning methods were attempted - Gibson assembly and PCR sewing with restriction enzymes. Gibson assembly failed to ligate correctly while PCR sewing excluded a small DNA segment but maintained the correct sequence otherwise.
3) Beta-galactosidase assays will be used to quantitatively measure protein expression of the introduced lacZ gene. Preliminary assays showed the method works for a shuttle plasmid and that the native methanogen has no background lacZ activity. The recombinant plasmid created
- ALK gene mutations can cause lymphomas such as ALCL and rare cases of DLBCL. The ALK gene encodes a tyrosine kinase receptor important for neural development. In ALCL, the ALK gene often fuses with other genes like NPM, forming chimeric proteins that drive uncontrolled cell growth. ALCL with ALK mutations has a better prognosis and responds well to chemotherapy, with many patients achieving long-term remission or cure. New targeted therapies that inhibit ALK are also under development.
Antibodies, also known as immunoglobulins, are glycoproteins produced by B cells that bind to specific antigens. There are five classes of immunoglobulins - IgG, IgM, IgD, IgA, and IgE - which differ in their heavy chains. Immunoglobulins have two primary functions: antigen binding and effector functions like complement fixation and binding to immune cells. Their structure consists of two light chains and two heavy chains that form a Y-shape, with constant and variable regions that determine the class and allow binding to different antigens.
The document discusses the generation of antibody diversity in the immune system. It explains that there are millions of possible antigens but only a small number of immunoglobulin genes in our genome. Through seven mechanisms, including multiple germline genes, combinatorial V(D)J joining, junctional flexibility, and somatic hypermutation, the immune system is able to generate a diverse repertoire of antibodies against all potential antigens from a limited set of gene segments. These mechanisms operate during B cell development and maturation in the bone marrow and lymphoid tissues.
The document summarizes the key mechanisms by which the human immune system generates a diverse repertoire of antibodies from a relatively small number of genes. It describes the somatic variation theory where mutation and recombination of immunoglobulin genes in somatic cells results in high antibody diversity. It explains processes like V(D)J recombination of light and heavy chain genes, junctional diversity, allelic exclusion, somatic hypermutation, and class switching which all contribute to antibody diversity.
The document provides an overview of apoptosis, or programmed cell death, discussing its molecular mechanisms and role in development and disease. It summarizes that apoptosis occurs through intrinsic and extrinsic pathways, is regulated by Bcl-2 family proteins like Bax and Bcl-2, and involves caspase activation leading to DNA fragmentation and phagocytosis of cell fragments. The document also discusses the importance of apoptosis in immune system development and its relevance to cancer.
(1) The study aimed to define the structure of the MLIP gene and its tissue-specific expression patterns.
(2) PCR and western blot experiments showed that MLIP exon 1a is expressed in heart and skeletal muscle, while exon 1b is expressed in the brain. Neither exon is expressed in the pancreas or liver.
(3) This suggests MLIP uses different promoters in different germ layers - exon 1a promoter in mesoderm, exon 1b promoter in ectoderm, and unknown promoters in endoderm tissues like pancreas and liver.
The prevalence of Copy Number Variation (CNV) on human chromosome 16p11.2, identified in approximately 1% of autism spectrum disorder (ASD) cases globally, was found to be 1.2% in an Australian ASD cohort. Bioinformatic analysis identified 13 of the 25 protein coding genes within the 16p11.2 region, including KCTD13, as significantly enriched in the nervous system. Experiments in mice found that suppression of Kctd13 led to impaired radial migration of cortical neurons and altered neuronal morphology, providing insight into how perturbations to KCTD13 expression via 16p11.2 microdeletion could influence brain development. Further experiments showed decreased cell proliferation and mitosis levels
This project aims to develop pharmacological agents that block T-type calcium channels (Cav3.2) for therapeutic applications. Rabbits were immunized with a peptide from the extracellular loop between transmembrane segments 3 and 4 of Cav3.2. Antibodies from rabbit serum showed immunoreactivity against the peptide. These antibodies will be tested for their ability to bind and block Cav3.2 channels expressed in HEK293 cells using techniques like western blotting and patch clamping. Successful blocking antibodies may aid in understanding Cav3.2 function and developing drugs targeting voltage-gated calcium channels.
Spherical nucleic acids (SNAs) can safely deliver nucleic acids to the body by binding to membrane proteins and being taken up by cells. SNAs can stimulate immunity by activating toll-like receptors (TLRs) on immune cells, producing inflammatory signals. The author aims to screen different oligonucleotide sequences on SNAs to identify those that strongly activate TLR9, using a Quanti-Blue assay to measure immune activation. Preliminary results show modified SNA sequences retain activity compared to the original sequence and elicit stronger responses than linear sequences.
This document summarizes an experiment to derive tissue culture cell lines that secrete antibodies against sheep red blood cells (SRBCs). Mouse myeloma cells were fused with spleen cells from mice immunized with SRBCs. The resulting hybrids were selected in HAT medium, and clones that secreted anti-SRBC antibodies were identified using plaque assays. Figures 1 and 2 show that the hybrids expressed antibodies from both parental lines and secreted antibodies that could lyse SRBCs. Figure 3 demonstrates antibody secretion patterns on isolectric focusing, while Figure 4 shows inhibition of lysis by anti-IgM antibody. This technique demonstrated the ability to produce predefined antibodies using fused myeloma-spleen cell lines.
- The naive follicular B-cell pool in mice lacking the surrogate light chain is enriched for B cells expressing antibody heavy chains with prototypic autoreactive CDR3 sequences.
- These mice spontaneously form germinal centers in the spleen that are enriched with B cells expressing prototypic autoreactive heavy chains.
- Peripheral tolerance is maintained by selection thresholds, as evidenced by the exclusion of intrinsically autoreactive VH81X-expressing B cells from the memory B-cell and plasma cell pools.
Mutations in the gene encoding Lamin B receptor (LBR), a nuclear-membrane protein with
sterol reductase activity, have been linked to rare human disorders. Phenotypes range from a
benign blood disorder, such as Pelger-Huet anomaly (PHA), affecting the morphology and
chromatin organization of white blood cells, to embryonic lethality as for Greenberg dysplasia
(GRBGD). Existing PHA mouse models do not fully recapitulate the human phenotypes,
hindering efforts to understand the molecular etiology of this disorder. Here we show, using
CRISPR/Cas-9 gene editing technology, that a 236bp N-terminal deletion in the mouse Lbr
gene, generating a protein missing the N-terminal domains of LBR, presents a superior model
of human PHA. Further, we address recent reports of a link between Lbr and defects in
X chromosome inactivation (XCI) and show that our mouse mutant displays minor
X chromosome inactivation defects that do not lead to any overt phenotypes in vivo. We
suggest that our N-terminal deletion model provides a valuable pre-clinical tool to
the research community and will aid in further understanding the etiology of PHA and the
diverse functions of LBR.
Human Leukocyte Antigen (HLA) typing involves determining the presence of HLA antigens on white blood cells. HLA antigens are encoded by genes in the major histocompatibility complex located on chromosome 6. HLA typing was originally done using serology to detect antibodies binding to HLA antigens, but now molecular techniques are more commonly used. HLA antigens are highly polymorphic and inherited as haplotypes from each parent, contributing to diversity in transplantation compatibility.
This grant proposal aims to analyze protein-protein interactions within the type-III secretion system (T3SS) of the pathogenic bacterium Chromobacterium violaceum. The researchers will use molecular techniques like PCR and the yeast two-hybrid system to study interactions between 11 proteins that are thought to be involved in Cpi-2, one of two T3SSs found in C. violaceum. Understanding these interactions could help identify new drug targets and vaccine components to treat infections caused by this antibiotic-resistant bacterium. The proposal outlines experiments to clone the genes of interest, test protein interactions using yeast two-hybrid screens, and build an interaction map of the Cpi-2 T3SS apparatus.
Cell communication allows cells to communicate through direct contact or chemical signals. Cell signaling involves cells detecting signals through receptors on their membranes, which leads to a response. A new discovery found that the PERK protein, known to detect protein folding errors, also coordinates communication between the inside and outside of cells. This opens possibilities for cancer, Alzheimer's, and diabetes research. PERK establishes contact between the endoplasmic reticulum and plasma membrane to help restore calcium levels and maintain cell functions under stress.
Nuclear Transport And Its Effect On Breast Cancer Tumor CellsStephanie Clark
The document discusses nuclear transport and its role in breast cancer tumor cells. It explains that nuclear transport involves the movement of large molecules in and out of the cell nucleus through nuclear pore complexes. It notes that various nuclear transport pathways play an important role in the progression and suppression of breast cancer tumor cells. Disrupting nuclear transport sequences can alter protein localization and functionality, as seen with spleen tyrosine kinase, which is a candidate tumor suppressor for breast cancer.
This document discusses several key concepts regarding antigens, antibodies, and the antigen-antibody interaction:
1) Antigens can be proteins, carbohydrates, nucleic acids, lipids, or small molecules. Antibodies bind to antigens through complementarity determining regions (CDRs) located in the variable regions of the heavy and light chains.
2) The structure of antibodies consists of two heavy chains and two light chains connected by disulfide bonds. Digestion with enzymes separates antibodies into fragments.
3) Antibody diversity arises from variations in the amino acid sequences of the CDRs. Mutations in the CDR regions allow each antibody clone to recognize a different epitope on an antigen
Oncogenic Ras promotes the survival of cancer cells detached from the extracellular matrix (ECM) through distinct downstream pathways. Ras activates phosphatidylinositol 3-kinase (PI3K) signaling to regulate metabolism in detached cells, but surprisingly does not do so through Akt. Instead, Ras utilizes serum and glucocorticoid-regulated kinase 1 (SGK1) to promote ATP generation. Additionally, Ras blocks anoikis or detachment-induced apoptosis by reducing expression of the phosphatase PHLPP1, which activates p38 MAPK to induce anoikis. Thus, Ras facilitates survival of detached cancer cells through divergent effectors to regulate metabolism and anoikis.
CELLULAR REPROGRAMMING: Current Technology, Perspectives and Generation of iP...Munna Yadav
Reprogramming refers to erasure and remodelling of epigenetic marks, such as DNA methylation, during mammalian development. Exposure of a differentiated cell nucleus to the cytoplasm of less differentiated cell leads to erasure of the stable epigenetic code that maintains the differentiated cell’s phenotype. Gradually, the nucleus acquires a new epigenetic code that is characteristic of the dedifferentiated cell donating the cytoplasm, a process termed cellular reprogramming.
This document summarizes a study that identified 24 splice variants of the human IL-23 receptor (IL-23R) gene from activated human leukocytes. The variants were characterized through restriction enzyme digestion and sequencing of IL-23R cDNA clones. Most variants involved exon skipping or deletions in the extracellular region of the IL-23Ra protein, which could impact IL-23 ligand binding and signaling. The identification of these potentially functional IL-23R variants improves understanding of how the gene contributes to both normal immune function and pathological conditions like inflammatory bowel disease.
This document discusses targeting brain endothelial cells for gene therapy to treat juvenile neuronal ceroid lipofuscinosis (JNCL). Researchers will inject adeno-associated virus (AAV) particles containing the CLN3 gene intravenously into JNCL mice. This is intended to deliver the CLN3 gene to endothelial cells lining the brain vasculature. Restoring the CLN3 gene in these cells may help them function properly and could prevent JNCL symptoms in mice. The mice will then be tested on motor tasks to see if their condition is improved compared to untreated JNCL mice. If successful, this approach could be advanced as a potential gene therapy for human JNCL patients.
This document summarizes three research studies on skin and keratinocytes:
1) The first study found elevated expression of osteopontin splice variants in nonmelanoma skin cancers compared to normal skin and adult keratinocytes. It also found that human adult keratinocytes expressed basal or induced levels of only two osteopontin variants.
2) The second study generated a mouse model lacking the desmoglein 1 protein and found it led to perinatal lethality and impaired skin barrier formation. Desmoglein 1 appears essential for normal epidermal morphogenesis.
3) The third study used fluorescence polarization microscopy and magnetic tweezers to investigate protein organization and force transmission at
1 At least 2 questions from this section will be on the .docxmercysuttle
1
At least 2 questions from this section will be on the final exam
SAMPLE QUESTIONS FOR THE FINAL EXAM
Question 1. Ferritin is a protein involved in the storage of iron inside cells. To prevent toxic accumulation of
too much iron inside cells, the intracellular level of ferritin is tightly regulated. To study the regulation of
ferritin synthesis, mammalian cells are grown with or without iron in the culture medium. Note that iron in the
culture medium is rapidly transported inside cells.
a) Upon addition of iron to the culture medium, the intracellular concentration of ferritin mRNA is unchanged
but the concentration of ferritin protein increases. How do you think ferritin expression is regulated? Briefly
explain.
The regulatory sequence given below is found in the ferritin mRNA between the cap structure and the start
codon.
5’-GGGUUUCCGUUCAACAGUGCUUGGACGGAAACCC-3’
Mutations within in this sequence are used to study the regulation of ferritin expression. The following
observation are made:
• ferritin expression is high, independent of the iron concentration, when (i) the entire region is deleted, or
(ii) the region located upstream of the underlined sequence is deleted or (iii) the underlined sequence is
replaced with a random sequence.
• ferritin expression remains iron-dependent when this region is replaced by the following sequence:
5’-GGGCUCAGGUUCAACAGUGCUUGGACCUGAGCCC-3’.
Note that the sequence differences are indicated in bold.
b) Explain why these observations suggest that both sequence and structure of the 5’ end of ferritin mRNA are
important for the regulation of ferritin expression.
c) Ferritin translation becomes iron-independent when the regulatory sequence is moved from the 5’ side
(upstream of the open reading frame) to the 3’ side (downstream of the open reading frame) of ferritin mRNA.
Which step of ferritin translation do you think is affected by the intracellular level of iron?
d) IRP is a protein involved in the regulation of ferritin expression. Anti-IRP antibodies attached to sepharose
beads are added to a cell extract, then the extract is centrifuged to separate the pellet fraction (containing the
sepharose beads ) from the supernatant fraction.
If the cells are cultured in the absence of iron, ferritin mRNA is found together with IRP in the pellet. In
contrast when cells are cultured in the presence of iron ferritin mRNA remains in the supernatant fraction while
IRP alone is found in the pellet. Briefly explain the likely role of IRP in the regulation of ferritin expression.
Question 2. You are studying the development of a newly discovered insect. Like drosophila, it undergoes a
stage in early larval development where the eve gene is expressed in a pattern of 7 stripes. You are particularly
interested in stripes 2 and 5. The following figures show the organization of the cis-acting elements that control
the expression o ...
1 At least 2 questions from this section will be on the .docx
FER poster
1. The Roles of ELL2 and ELL3 in Plasma Cell Development
Shane McKeon and Aleksandra Basina Christine Milcarek
First Experiences in Research, Dietrich School of Arts & Sciences, University of Pittsburgh
Abstract
The Lenti Virus
DiscussionIdentifying ELL2 and ELL3
Acknowledgements
B cells express immunoglobulin (Ig) molecules composed of heavy and light chains on their surfaces; after
antigen or LPS stimulation they differentiate into antibody secreting plasma cells (ASCs) that make and secrete large
amounts of the Ig protein. One of the primary processes to influence the shift to Ig secretion is differential Ig heavy
chain RNA polyadenylation and splicing. We showed in unpublished data that the small nuclear RNAs important for
splicing are reduced in ASCs relative to B cells (Carew & Smith, personal communication). Previous studies showed
that (a.) the transcription elongation factor ELL2 is induced with differentiation to ASCs, (b.) conditional deletion of
ELL2 results in decreased Ig heavy chain secretory-specific mRNA production and diminished Ig secretion in the whole
animal and (c.) after LPS stimulation of B cells ELL3 decreases and ELL1 is unchanged (1). The ELL3 and ELL1 and
2 proteins are closely related in structure but ELL3 lacks the interior ~100 amino acids (2). The ELL proteins have been
shown to be involved in at least two transcription elongation complexes, the little elongation complex (LEC) involved
with small nuclear RNAs, and the super elongation complex (SEC) important for the bulk of genes transcribed in
Drosophila and human cancer cell lines (3). However, the differential involvement of ELL2 versus ELL3 was not
explored. We then hypothesized that the transition in expression of ELL3 to ELL2 is important in the development of B
cells into ASCs, and would influence both Ig secretory mRNA expression and small RNA synthesis. The Milcarek
Laboratory is using Quantitative PCR (QPCR), western blots and complementation of defects in knockout mice to
display the relationships. The research this semester was primarily focused on seeing whether or not ELL1, 2 and 3 had
similar functions. To do so, we first needed to test our cell lines to make sure we had the correct antibodies, and grow a
virus that could be used to correct deficiencies in the immunoglobulin secretion abilities of splenic B cells from ELL2
conditional knockout mice.
We began with two cell lines, MM1.S containing only ELL2, and SKW6.4 containing only ELL3. In order to
provide concrete support that these cells line expressed the desired proteins we ran a QPCR which showed that SKW6.4
does indeed contain ELL3 while MM1.S does not (Figure 1). Furthermore, a western blot was run using antibodies
complimentary to ELL2. Once the blot was transferred to a membrane and imaged with chemiluminescence, it was clear
that MM1.S contains ELL2, while SKW6.4 did not. (Figure 2)
Moving forward, Lenti virus DNA will be packaged and placed into knockout mice cells (contains no ELL2) and wild
type mice cells (contains ELL2). The cells without ELL2 should produce no immunoglobulin secretion as their immune
system has been compromised. ELL1, ELL2 and ELL3 will then be individually added to the knockout cells. It is
expected that ELL2 will correct this defect, however, the main purpose of this experiment is to see if ELL1 and/or ELL3
will also correct the defect. If so, it will prove that ELL1 and/or ELL3 have similar if not the same function within the
immune system. Future directions will involve characterizations of the LECs and SECs for B versus ASCs.
We would like to acknowledge Christine Milcarek for guiding us through this research and supporting our
love for research. Also we would like to thank FER for this experience.
Background Information
When B cells differentiate into antibody secreting cells many genes are
affected but little information is known. In a previous study, Dr.
Milcarek found that there were changes in the expression of small
nuclear RNA; an important component in splicing out introns from pre-
RNA to form mature mRNA. Additionally, it was found that ELL3
levels are diminished after stimulation from B cells to ASC. As seen in
the figure to the right, ELL3 is much lower in ASCs than B cells, while
ELL2 is much lower in B cells than ASCs. This information led to
looking into how snRNA is controlled during the transition from B cells
to ASCs.
It was hypothesized that ELL3 regulates the formation of SECs and
LECs in B cells while ELL2 drives the formation of the SECs over the
LECs when producing antibodies. It is this switch that benefits mRNA
instead of snRNA.
To test the hypothesis, we wanted to see if the down regulation of snRNA was
linked to the down regulation of ELL3. Naïve splenic cells will be stimulated
with LPS and then treated with inhibitors of protein synthesis or RNA
synthesis and the production of ELL3 and snRNA will be monitored. Prior to
doing so, the research mainly focused on identifying which cell lines contained
ELL2 vs ELL3 so that we could be sure we had the correct genes.
Additionally, we decided to grow a lenti virus that would be placed into ELL2
knockout splenic B cells. We would then inject the ELL proteins one at a time
and see if one, or all could correct the deficiency, by monitoring the
immunoglobulin secretion.
The image to the left shows the similarities and differences between all three
ELL proteins on a molecular level. As you can see most of ELL3’s DNA
sequence is unknown and the parts that are shown are similar to the other ELL
genes, suggesting a similarity between the genes functions.
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To obtain a virus to transfect into the splenic B cells we ordered bacteria that
contained a lenti virus, but needed to test if the sample actually contained the
desired virus. The sample came with a plasmid map (as seen to the left) , that
showed what enzymes would cut the plasmid, and at what locations. For our
purposes we choose three enzymes, EcoR1, Sac1 and Sal1. These enzymes
were predicted to produce segments of the plasmid at 6973 and 3401 for
EcoR1, 2960, 191, 7223 for Sac1 and 1618, 2185, 6571 for Sal1. To test if our
lenti virus would be cut in the appropriate locations, we mixed the enzymes
with their complementary buffers and the DNA and ran each enzyme on a DNA
gel , with three different samples of the lenti virus. In the gels below, lanes 6,
12, and 16 contain the lenti virus uncut, so serve as a control.
Lanes 7, 13, and 17 contain the lenti virus cut
with the EcoR1 enzyme and came out to be
around 6000 kDa, as expected.
Lanes 8, 14,and 18 contain the Sac1 enzyme and
as seen in lanes 14 and 18, two bands appeared,
one around 7000 kDa and another around 3000
kDa. Additionally, lane 8 contains the band at
7000 kDa and another very faint band around
200 kDa. All three of these bands were expected
when cutting the plasmid with sac1.
Lanes 9, 15 and 19 contain the lentu virus cut
with Sal1 and all three lanes contain three bands
around the 6000 kDa mark, the 2000 kDa and
the 1000 kDa mark. All three values are
consistent with the predicted values provided by
the plasmid map.
In order to confirm that the MM1.S cell line contained ELL2 while the SKW6.4
cell line did not, we ran a protein gel and western blot. Because the MM1.S cells
were ASCs we expected a much large abundance in ELL2 than in the B cells
(SKW6.4). In order to test the results the gel was transferred to a western blot and
coated with a primary antibody that was complementary for ELL2 overnight. It
was then washed and a anti- ELL2 secondary antibody was placed on the
membrane. We were then able to expose the membrane to chemiluminescence
and received the image to the left.
The expected molecular weight of ELL2 is 72 kDa. As seen in the image there
are thick bands within the ASC lanes that are near 72 kDa, as seen on the protein
ladder next to the membrane image. Additionally, there are no bands around 72
kDa under the B cell lanes, proving that ELL2 is expressed in ASCs but not B
cells.
PCR is typically used to amplify a specific gene, or portion of gene, so that we can
study the function of that gene or gene region. In our experiments, we amplify the
lentiviral DNA that contains a vector with the gene eGFP (green fluorescent protein)
as well as ELL3. Primers are used to flank the region you want to amplify. Each
primer will amplify the gene sequence on both strands, creating a double-stranded
gene product. The primers we used are represented at the figure below.
The PCR process follows 3 steps. Denaturation step: First, you heat the DNA to a
high temperature (95 °C) so that the two strands of genomic DNA, and later PCR
DNA, separate. Annealing step: Second, you reduce the temperature (around 60 °C)
so that DNA primers bind to either end of the template that you want to amplify. It
is important that you have two primers, one to bind
to each strand of DNA. Extension Step: Third, you
raise the temperature to about 70 °C to activate a
DNA polymerase and elongate the primer with
respect to the template strand.
Over the course of the semester in the Milcarek lab, we were
ultimately able to lay the ground work for further testing of whether
genes ELL1 and ELL3 have similar function in the immune response
to that of the gene ELL2.
We were able to ensure that the MM1.S cell line contained only
ELL2 and the SKW6.4 cell line contained only ELL3 through
quantitative PCR analysis as well as western blots using antibodies
known to be complimentary to ELL2 (and therefore able to detect its
presence). In addition, we were able to confirm that the lenti viral
vector containing our genes of interest were present in the bacterial
host, and therefore could be used to transfect into B cells of
conditional knockout mice.
References
1. Park, K. S., Bayles, I., Szlachta-McGinn, A., Paul, J., Boiko, J., Santos, P., Liu, J., Wang, Z., Borghesi, L.,
and Milcarek, C. (2014) J. Immunol. 193, 4663-4674
2. Miller, T., Williams, K., Johnstone, R. W., and Shilatifard, A. (2000) J. Biol. Chem. 275, 32052-32056
3. Luo, Z., Lin, C., and Shilatifard, A. (2012) Nat Rev Mol Cell Biol 13, 543-547
4. Sage M. Smith, Nolan T. Carew, and Christine Milcarek Department of Immunology, University of
Pittsburgh, School of Medicine, Pittsburgh, PA 15261, United States
Recall that B cells differentiate into antibody secreting plasma cells (ASCs) upon stimulation by an antigen or
lipopolysaccharide (LPS). ASCs make and secrete vast amounts of immunoglobulin molecules (Ig), and that this
is influenced by differential IG heavy chain RNA polyadenylation and splicing.
Also recall that ELL2 is the only gene of the three ELL genes that has previously been shown to act directly on
immunoglobulin genes. It has so far been established that ELL2 is induced at least 10-fold in antibody secreting
cells in comparison to B cells, while ELL1 expression decreases slightly and ELL3 expression decreases
tremendously. In turn, such changes cause the RNA polymerase II in antibody secreting cells to take on different
properties, ultimately resulting in changes concerning RNA processing and modification of histones (4).
Continued research will focus on the role of ELL1 and ELL3 and whether they may substitute ELL2 in such
functions.
In the future, the lenti virus containing the gene of
interest will be transfected into cells of conditional
knockout mice. This will test whether ELL1 and/or
ELL3 can have similar functionality as that
exhibited by ELL2. In addition, further research
may lead to characterization and analysis of the
long elongation complexes (LECs) and the short
elongation complexes (SECs) present in B cells as
opposed to in ASCs.