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This document provides an overview of topics covered in a study guide for a biosignaling and gene regulation course. It includes summaries of key concepts and figures related to: - Signal transduction pathways such as cAMP/PKA, G protein coupled receptors, insulin and JAK-STAT signaling. - The basic structure and functions of nucleosomes, histone variants and chromatin remodeling complexes in gene expression regulation. - Important elements and processes in the lytic and lysogenic cycles of bacteriophage lambda. - Core concepts and techniques in recombinant DNA technology including restriction mapping, Southern blotting, oligonucleotide synthesis, chromosome walking, and use of yeast artificial chromosomes. - Specialized biotechnology topics such

48005/A8005 Study Guide unit 2 Spring 2022
Biosignaling
-Scatchard analysis Box 12-1 p.421
- -4, p.424
-cAMP activation of PKA figure 12-6, and epinephrine cascade figure 12-7,
p.429
-desensitization of -8, p.430
-receptor-coupled activation of phospholipase C figure 12-10, p.433
-insulin receptor/MAP kinase cascade figure 12-15, p.441
-GSK3 activation activation by insulin figure 12-16, p.442
-JAK-STAT pathway figure 12-18, p.443
-guanylyl cyclase coupled receptors figure 12-20, discussion pp.445-446
-visual system/light receptors pp 462-465, figures 12-35, 12-36, 12-37, 12-38
-olfaction and gustatory signaling figures 12-40, 12-41, pp.465-468
Regulation of gene expression -
-Go over the basic structure of nuclesomes from Lehninger as covered in
class:
the nucleosome subunits (H1, H2A, etc)
the structure and usage of the SMC proteins
know the basic role/functions of: condensins, cohesins, separase,
histone variants, CAF1, RCAF, ASF1
-The activities of SAGA/PCAF, NuA4, SWI/SNF, ISWI, HIRA (Table 28-2)
-For regulation of gene expression go over the gene expression paradigms
shown in figs 28-29,28-30,28-31, 28-32,28-33, 28-34.
Bacteriophage Lambda -
Function of elements in table 32-1 in the handout as covered in class. Be
able to identify their function in the lytic cycle or lysogeny? For example
"Xrz blocks transcription from U' by binding to the Y activator. As a result,
the bacteriophage is unable to carry out the RnR function that requires
expression of genes controlled by the Z promoter."]. Study the diagrams
covered in class (figures 33-29, 33-31, 33-40, 33-43, 33-47, 33-49, 33-50,
33-51, 33-52 in the handout).
List of important elements of the lambda genome:
cI
cII, cIII
cro
N,Q
O,P
int
xis
R,R2,S

attP, attB
cos
OL, OR
PI,PL,PR,PRM,PRE R
nut L, nut R
qut
rho
hflA, hflB
Recombinant DNA technology
-Go over restriction enzymes and the basic principles of cloning in plasmids,
cosmids, bacteriophage, and YACs in Lehninger chapter 9 (DNA-based
information technologies)
In the Steinberg handout:
Section 1.4.2 restriction enzymes. Particularly go over the
restriction mapping example shown in the figure on handout page 35
Understand the principle of Southern blotting shown in Figure 1.8
Particularly understand the RFLP example in Figure 1.9 using sickling
hemoglobin (pp 12-15).
Know the basic steps in oligonucleotide synthesis shown Figure 1.13
Know the steps in the process and the basic chemical modifying groups
(DMT, cyanoethyl-, alkyamine chain, diisopropyl amine, etc.) and
reagents.
Understand the principles of chromosome walking described in Section
1.4.9
The elements of Yeast artificial chromosomes (YACs) in section 1.4.13
of the biotechnology handout
-Go over the specialized topics in biotechnology:
real time PCR
microarrays
the yeast two hybrid system
Next generation sequencing
Crispr cas9 gene editing
Knockout-knockin mice
It will be helpful to go over the figures in the handouts and those shown on
the designated websites (the list of websites will be posted on blackboard).

BIOC 405 Assignment 1: Dr Moore Due Friday March 1st, 2019 before 16:00 in Room 3D30.8 HSc 1. (a)In your handout for protein kinase A, there is a table of known substrate sequences, in other words sequences of peptides phosphorylated by PKA. Please do your best to align the substrate sequences provided, and from the alignment, predict what a good consensus substrate for PKA will be. To present your alignment, please use an equal width font for the protein sequences (Courier or Courier New work well). Highlight the P(0) residue, P(-1) etc. (b)The regulatory subunit (R) of protein kinase A has a short sequence (RRRRGAISA that is critical for inhibiting the activity of the kinase catalytic subunit. This short sequence of the R-subunit actually sits in the active site cleft of PKA in the crystallographically-determined structure of the inhibited RC complex. Can you deduce what the function of this sequence is? Using the answer to part (a) as a guide, please align the inhibitory sequence with the known substrate sequences to deduce how this sequence likely functions to inhibit PKA. Furthermore, using your class notes, can you make a guess at which residues on PKA might interact with specific residues from the R-inhibitory peptide? 2. For a regularly spaced 1-Dimensional array of atoms (spacing =13 Å) calculate the total number of diffraction maxima and their scattering angles (for perfect in phase scattering from the atoms in the 1-D array) between scattering angles of zero and ninety degrees Use a wavelength of d=1.25 Å. Please include a drawing to explain the diffraction condition and show your calculations. 3. Using site specific mutagenesis to change residues in the substrate binding cleft of PKA (not residues involved in catalytic roles), how would you alter PKA’s substrate specificity at P-3, and P-2 to Glu and P+1 to Asn? By this, I mean how would you specifically make mutations in the PKA enzyme amino acid sequence (not the substrate sequence) that would select for binding and phosphorylation of a peptide sequence that would clearly differ from the known substrate sequence preferred by PKA as outlined above. Be sure to clearly highlight exactly what residues in the PKA sequence you would have to change (and to what amino acid) to achieve this. 4. The following lines of data describe the atomic coordinates for an arginine residue in a protein molecule in PDB (protein data bank) format. Since proteins are three-dimensional objects, the position of each atom is specified in space by its X, Y and Z coordinates. On each line of a PDB formatted file, the atom number is given, the atom type is next (e.g. N- backbone nitrogen, backbone carbonyl oxygen etc), the residue name (here ARG 431 in chain D; in this instance the protein crystal contains four independent copies of the polypeptide chain, labelled A through D), then the X-coordinate for that atom, the Y-coordinate for that atom and the Z-coo.

Cytoscape: Integrating biological networks

BITS

Li, Lisanti, Puszkin - Purification and molecular characterization of NP185

Shengwen Calvin Li, PhD

This study further characterized the neuronal protein NP185/AP3 through several experiments: 1) Microsequence analysis of purified native NP185 from bovine brain yielded five peptide fragments that matched sequences of mouse F1-20 and rat AP180 proteins. 2) Recombinant NP185/AP3 purified using genetic methods facilitated the reconstruction of clathrin cages in an established assay. 3) Deletion mutagenesis mapped the epitopes of two monoclonal antibodies that recognize bovine NP185 to a 60 amino acid region of recombinant murine NP185/AP3. 4) Recombinant NP185/AP3 was phosphorylated by purified casein kinase II in vitro.

GRAS proteins expression and purification

Mesele Tilahun

The document summarizes the production and analysis of the GRAS protein Os-SCL7. It describes how the gene encoding the GRAS domain of Os-SCL7 was cloned and expressed in E. coli. The protein was then purified using nickel affinity chromatography and size exclusion chromatography. Sequence analysis revealed the protein is 378 amino acids with a predicted molecular weight of 41.5 kDa. Potential cleavage sites for specific proteases were also identified from the amino acid sequence.

Differential metabolic activity and discovery of therapeutic targets using su...

Joaquin Dopazo

The document discusses obtaining estimations of cell metabolic activities from gene expression data using summarized metabolic pathway models. It describes normalizing gene expression data and using an algorithm to estimate metabolic module activities for two conditions, identifying differentially active modules using statistical tests. Metabolic module activities are correlated with protein activation status and metabolite abundance changes, associated with cancer types and treatment responses, and predictive of cell survival. A web server called Metabolizer is introduced that performs various metabolic pathway analyses on gene expression data.

Structural Mechanism for the Fidelity Modulation of DNA Polymerase λ

Mu-Sen Liu

The document summarizes research on the structural mechanism that allows DNA polymerase λ (Pol λ) to achieve medium fidelity. Key findings include: 1) Pol λ can bind nucleotide substrates (MgdNTPs) selectively in the absence of DNA, with highest affinity for MgdATP. 2) The crystal structure of apo Pol λ surprisingly exists in a closed conformation, like its DNA-bound form, with the nucleotide binding pocket already preformed. 3) Mutations that enhance MgdNTP prebinding (e.g. L431A) lower the enzyme's fidelity, indicating prebinding ability relates to its medium fidelity level. The structures provide insight into Pol λ

Regulation of pten activity by its carboxyl terminal autoinhibitory

Chau Chan Lao

Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain. Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007 目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白，其由403個氨基酸組成，主要分PTPase及C2 domain，C2 domain使PTEN可與細胞膜作用連結。 PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385)，這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株，發現這些變異株具有更強的腫瘤抑制能力，但穩定性將降低，這可能是因這些變異株具有更開放結構所致。本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株，發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro，而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低，因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法，發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用，暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide，發現其可抑制PTEN之催化活性in vitro，而在細胞表現此peptide則會抑制PTEN之membrane localization，磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。

The document discusses using surface entropy reduction (SER) mutations to improve crystallization of an RNA-Fab complex. Key points: - SER mutations were identified on the Fab surface using software to reduce flexibility and promote crystal contacts. Mutations were incorporated using Kunkel mutagenesis. - Mutant Fabs (Fab2SMA, Fab2SMS) were expressed at 3-4 mg/L and bound RNA similarly to the wild type Fab. Initial crystal hits were found for the mutant complexes. - The SER approach aims to generalize to other RNA-Fab complexes, as demonstrated by binding of mutant Fabs to the VCIII riboswitch. Optimization of crystal hits is ongoing to improve diffraction resolution

Kieran_ccpNMR

Kieran Hand

This study investigated the phosphorylation of α-synuclein (α-Syn), a protein implicated in Parkinson's disease, using NMR spectroscopy. α-Syn has three domains and adopts different structural assemblies during its aggregation process. Phosphorylation may contribute to α-Syn aggregation, though the mechanisms are unclear. The study used NMR to monitor the activity of CK2α, a kinase known to phosphorylate α-Syn at Ser129. Preliminary results found CK2α phosphorylated α-Syn as observed by biochemical analysis and minor chemical shift changes in NMR spectra. Future work will further characterize the effects of phosphorylation on α-Syn aggregation in both buffer and cell lysates using techniques like NMR, fluorescence,

The invention of sprycel from benchtop to bedside (Gengcheng Jack Yang)

Jack Gengcheng YANG

1) The document describes the discovery and optimization of dasatinib (BMS-354825), a potent pan-Src kinase inhibitor. Initial screening identified a thiazole-based hit that inhibited Lck kinase. Extensive SAR explored substituents on the thiazole ring as well as replacements for the amide linker. 2) Replacing the amide with a 2-aminopyrimidine improved potency against Lck and cellular assays. Further appending a polar side chain to this analog enhanced aqueous solubility and potency, yielding dasatinib. 3) Dasatinib was found to be a highly potent, selective, ATP-competitive inhibitor of Src family kinases and

Quantitative In silico analysis of mammalian serine racemase activity

AI Publications

Serine racemase (SR) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme catalyzing the racemization of S-serine to R-serine, and also oxidizes R-serine to pyruvate; however, the enzymatic racemization reaction is not fully understood. Therefore, quantitative in silico analysis of mammalian SR was performed followed byD-amino acid oxidase (DAO) analysis.The downloaded stereo structures of Rattus norvegicus (PBD ID: 3hmk) and mammalian (PDB ID: 3l6r and 3l6b) SR were optimized using molecular mechanic (MM2) calculation. Furthermore, the stereo structure of mammalian SR was constructed using 3hmk and the amino acid sequence data. The atomic partial charge (apc) of the target atoms was calculated using MOPAC-PM5. The atomic distances, bond angles, and apc of the mutants were used to study the enzyme reaction mechanism.The serine carboxyl group contacted lysine-56, where its amino group contacted the oxygen molecules of PLP aldehyde. The apc of C indicated that S-serine was selectively oxidized. Manganese located at another side of PLP is directly involved in the serine racemization by forming bonding with the hydroxyl group of serine. The enzyme racemization activity was quantitatively related to the bond angle of the substituted serine rather than to that of alanine. However, 316r and 316b lack several amino acid residues; therefore, the selective oxidation of S84 mutants was not clearly demonstrated.

Structural Modeling of the Ptf1a C2 Sequence Bound to Mammalian Rbpj and Rbjl

Ward Coats

In this study, the researchers: 1) Generated structural models of the C2 sequence of Ptf1a bound to the β-trefoil domains of mammalian Rbpj and Rbpjl. 2) Found that the C2 sequence binds in an extended conformation, maintaining interactions seen in the Notch-IC/Rbpj complex. 3) Observed conserved residues in the hydrophobic pocket that maintain binding of proteins with a conserved tryptophan motif.

Honors Biology -Molecular Biology

Michael Edgar

The document discusses several topics related to DNA replication, genetic engineering, and gene regulation: 1) It describes the basic process of DNA replication, including the roles of helicase, primase, topoisomerase, and other proteins. 2) It explains some techniques used in genetic engineering, such as restriction enzymes cutting DNA at specific sites, ligation to join DNA fragments, and gel electrophoresis to separate DNA fragments by size. 3) It discusses mechanisms of gene regulation, including repression and activation of operons in response to metabolites and how this controls enzyme production for metabolic pathways like tryptophan and lactose synthesis.

Small Molecule Interactions with Protein Tyrosine Phosphatase

Jonathan Paul

This document summarizes the results of ligand docking experiments with Protein Tyrosine Phosphatase 1B (PTP-1B). 63 ligands were docked to various crystal structures of PTP-1B, including wild type and mutant variants. The top two ligands, 2CNF and 1Q6S, docked with scores of -9.512 and -9.433 respectively to the wild type 1PXH structure. Common interacting residues for ligands included Arg221, Ser216, and Gln262. Future work could include expanding the ligand library and performing quantitative structure-activity relationship (QSAR) studies to help optimize ligands for inhibiting PTP-1B.

Summer 2015 REU Poster AK_FINAL

Andrew Kocian

RNase contamination was preventing accurate measurement of RNA-protein binding between LARP6 and its RNA substrate. The researchers optimized native polyacrylamide gel electrophoresis with biotinylated RNA to visualize and quantify binding. They showed biotinylation worked without detecting RNase contamination. Continued gel condition optimization will improve resolution for determining the functional boundaries of the LARP6 La Module domain required for RNA binding.

2016 RBC RETREAT POSTER TEMPLATE_SMP

Sagar M. Patel

- The study characterized the kinetic properties of wild-type human Δ1-pyrroline-5-carboxylate reductase 2 (HsPYCR2) and its R251C mutant found in patients. - Kinetic analysis showed the R251C mutant had slightly lower catalytic efficiency (4-5 fold) for NADH and NADPH substrates compared to wild-type, suggesting the mutation impacts proline biosynthesis. - Additional studies on protein structure may help understand how the R251C mutation contributes to neurological disease phenotypes seen in patients.

Signal transduction processes connected to the changes in cytosolic calcium c...

Pradeep Singh Narwat

Calcium signalling occurs due to changes in cytosolic calcium concentration and activates protein kinase C. Protein kinase C is activated when phospholipase C breaks down phospholipids like phosphatidylinositol, producing diacylglycerol which remains in the cell membrane and recruits protein kinase C to the membrane. This allows calcium-calmodulin complexes to activate protein kinase C, leading to phosphorylation of transcription factors and regulation of processes like cell growth and metabolism. Phospholipase C also produces IP3 which triggers release of calcium from intracellular stores, further increasing cytosolic calcium levels.

Cholesteric Liquid-Crystal Copolyester, Poly[oxycarbonyl- 1,4-phenylene- oxy ...

IJERA Editor

The cholesteric liquid-crystal poly[oxycarbonyl-1,4-phenylene-oxy-1,4 terephthaloyl-oxy-1,4-phenylenecarbonyloxy( 1,2-dodecane)] [C34H36O8]n, named PTOBDME, synthesized by polycondensation reaction from equimolar quantities of TOBC and the racemic mixture of glycol (R-S-1,2 dodecanediol), exhibits unexpected optical activity and chiral morphology. The structure of racemic-PTOBDME, under different polymerization kinetics conditions, is analyzed by conventional NMR techniques and compared with those of polymer enantiomers R-PTOBDME and S-PTOBDME obtained starting R(+)1,2 and S(-)1,2-dodecanediol respectively. Molecular models based on the NMR signals intensities are proposed. The optical activity of racemic- PTOBDME is evaluated by measuring the ORD values during kinetics study, and compared to the chiral polymers. Each enantiomeric polymer seems to present the same stereoregular head-tail, isotactic structure than the racemic, which we explain by the higher reactivity of the primary hydroxyl than the secondary one in the glycol through polycondensation. For each enantiomer, two independent sets of signals were observed by NMR, explained as two diastereomeric helical conformers: gg and gt, related with two possible staggered conformations, along the copolymer backbone. Chirality in racemic-PTOBDME is proposed to be due to the kinetic resolution of a preferable helical diastereomer, such as Sgt, with respect to the possible four forms, while the R/S ratio of asymmetric carbon atoms remained 50:50. Chiral amplification is observed in R-PTOBDME and S-PTOBDME due to a helical screw sense excess. Optimum yield was obtained for racemic PTOBDME, after 120 minutes polycondensated and decanted in toluene for 24 hours. Two weeks later a second fraction precipitated from the toluene mother liquor with 67.6% chiral excess. After eight months and two weeks a third fraction precipitated with 85.2% chiral excess.

NAPP-DR6 poster

Kelly Considine

This study used computational methods to identify peptide inhibitors of the interaction between the death receptor DR6 and the N-terminal fragment of amyloid precursor protein (NAPP), which is implicated in Alzheimer's disease (AD). A focused peptide library was virtually screened and peptides were docked and scored against the NAPP binding site. Several tetrapeptides were predicted to bind strongly to NAPP, particularly Pro-Lys-Trp-Trp, which was predicted to inhibit the DR6-NAPP interaction through hydrogen bonding with key NAPP residues. Future work will include synthesizing and testing the top peptides to validate the computational predictions.

P68 RNA helicase unwinds the human let-7 microRNA precursor duplex and is req...

David W. Salzman

P68 RNA helicase was identified as being required for unwinding the human let-7 microRNA precursor duplex. Recombinant P68 RNA helicase was shown to unwind the let-7 duplex in vitro. Knockdown of P68 inhibited let-7 microRNA function, indicating P68 is essential for loading let-7 into the silencing complex. This study identifies P68 RNA helicase as playing a key role in the human let-7 microRNA pathway.

ketoartic_le .pdf

AleenaKhan778846

Human Aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20 ketosteroid reductase with a preference for the conversion of Δ4-androstene-3,17- dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform-specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform-specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis. Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9α,11β-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARγ). Activation of PPARγ is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9α,11β- PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti- inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and 9α,11β-PGF2. Structure-activity relationships Thirty-five crystal structures of AKR1C3·NADP + ·inhibitor complexes exist in the PDB. Inspection of these structures shows that if the inhibitor contains a carboxylic acid, it can often form hydrogen bonds with the catalytic tetrad members Tyr55 and His117. Other portions of the inhibitor can occupy one of several subpockets (SP), e.g. SP1 Ser118, Asn167, Phe306, Phe311, and Tyr319 (e.g. occupied by the B-ring of N- cphenylaminobenzoates). The SP2 sub-pocket refers to Ser129, W227, and F311 (e.g. occupied by the side-chain of PGs), and the SP3 sub-pocket which contains Y24, E192, S217, S221, Q222, Y305, and F306. While the presence of these sub-pockets can be rationalized to determine the binding mode and can be used as the basis of docking studies,

Characterization in Dvilp 7 gene

Hunter Kelley

This study characterized the Dvilp7 gene from Drosophila virilis through a series of experiments. RNA was purified from D. virilis and used to construct cDNA. RACE experiments were used to amplify the 5' and 3' ends of the Dvilp7 cDNA sequence. The full Dvilp7 cDNA sequence was assembled and found to encode a putative protein with a signal peptide. Genomic DNA was also sequenced and compared to determine intron sequences. Characterizing the Dvilp7 gene expands understanding of the genetic mechanisms regulating insulin signaling in Drosophila.

MOLSTRUC Unveiling Structural Features, Chemical Reactivity, and Bioactivity ...

Trustlife

Fall 2015 Sitag Poster _120516 (1)-2

Adam Hildebrandt

1. Researchers developed a Si-tag fusion protein containing Si-tag, RGD, and His-tag to enable delivery of biomolecules like RGD onto silica surfaces for tissue engineering applications. 2. They expressed the fusion protein in E. coli and tested various purification methods, finding that rapid purification using silica nanoparticles was more effective than silica gel or IMAC. 3. Functionality assays using immunostaining and binding to silica nanoparticles confirmed the fusion protein retained its ability to bind silica surfaces. Further optimization of purification conditions is still needed to improve protein yield.

Honors ~ DNA 1011

Michael Edgar

The document discusses several topics related to DNA and genetics including DNA replication, restriction enzymes, gel electrophoresis, PCR, DNA sequencing, cloning, and gene expression. It provides figures to illustrate key concepts and techniques such as how restriction enzymes cut DNA, how gel electrophoresis separates DNA fragments by size, how PCR amplifies a target DNA sequence, and the process of cloning a gene into a bacterial plasmid.

Final_Randhawa

Samender Randhawa

The document summarizes research characterizing the radical S-adenosyl-L-methionine (SAM) epimerase NeoN. NeoN catalyzes the specific epimerization of carbon C-5''' in neomycin C to produce neomycin B. The goal is to determine NeoN's structure using X-ray crystallography to understand its catalytic activity. Initial experiments cloning the neoN gene from Streptomyces fradiae and expressing the protein are presented, though yields were low. Future work will optimize cloning and crystallization to elucidate NeoN's structure and characterize residues involved in substrate recognition.

JACS-CD38 localization using a fluorescent probe

Jonathan Shrimp

This document describes the development of a fluorescent small molecule probe called SR101−F-araNMN that can label the enzyme CD38 in live cells in a mechanism-based manner. The probe was used to investigate the cellular localization of CD38 in leukemia (HL-60 and K562) and lymphoma (Raji) cell lines. The results showed that CD38 is predominantly localized to the plasma membrane in Raji and RA-treated HL-60 cells, with very little intracellular CD38. Additionally, no CD38 expression was detected in K562 cells. This suggests that the major function of CD38 is to hydrolyze extracellular rather than intracellular NAD.

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