This work was supported by grants from the National Institutes of Health (HL-141630 and HL-160569) and Cincinnati Children&#39;s Research Foundation (Trustee Award).

The protocol outlined below details steps to load and navigate the PhyloCSF browser tracks on the UCSC Genome Browser (generated by Mudge et al.49). For general questions regarding the UCSC Genome Browser, an extensive Genome Browser User&#39;s Guide can be found here:&#160;<a href="https://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html">https://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html</a>.
1. Loading the PhyloCSF Track Hub to the UCSC Genome Browser
<ol>
	<li>Open an internet browser window and navigate to the UCSC Genome Browser (<a href="https://genome.ucsc.edu/">https://genome.ucsc.edu/</a>).</li>
	<li>Under the Our tools heading, select the Track Hubs option. 
	NOTE: The Track Hubs option can also be found under the My Data tab.</li>
	<li>In the Public Hubs tab, type PhyloCSF into the Search terms box. Click on the Search Public Hubs button.</li>
	<li>Connect to PhyloCSF by clicking on the Connect button for the Hub Name PhyloCSF (Description: Evolutionary protein-coding potential as measured by PhyloCSF). 
	&#8203;NOTE: This Track Hub will load to numerous assemblies, including human (hg19 and hg38) and mouse (mm10 and mm39).</li>
	<li>After clicking on connect, wait to be redirected to the UCSC Genome Browser Gateway page (<a href="https://genome.ucsc.edu/cgi-bin/hgGateway">https://genome.ucsc.edu/cgi-bin/hgGateway</a>).</li>
</ol>
2. Navigating to genes of interest using Gene Identifiers
<ol>
	<li>Select the species and genome assembly to query. To query a different species (e.g., mouse), select the species of interest under the Browse/Select Species heading by clicking on the appropriate icon, or type the species into the text box that says, Enter species, common name or assembly ID. 
	NOTE: The assembly is listed directly under the Find Position heading. Typically, the default is the Human Assembly (e.g., Dec. 2009 [GRCh37/hg19]).</li>
	<li>Choose the assembly to search under the Find Position heading using the dropdown menu.</li>
	<li>Enter the position, gene symbol, or search terms in the Position/Search Term box and click on Go to navigate to a gene of interest on the Genome Browser.</li>
	<li>If the search resulted in multiple matches, wait to be redirected to a page that requires the selection of a position of interest. Click on the appropriate gene of interest.</li>
</ol>
3. Navigating to genomic regions of interest using sequence information
<ol>
	<li>Navigate to the UCSC Genome Browser (<a href="https://genome.ucsc.edu/">https://genome.ucsc.edu/</a>) and select the BLAST-Like Alignment Tool (BLAT) under the&#160;Our tools heading to query a specific DNA or protein sequence. Alternatively, hover the cursor over the Tools tab and select the Blat option or follow this link:&#160;<a href="https://genome.ucsc.edu/cgi-bin/hgBlat">https://genome.ucsc.edu/cgi-bin/hgBlat</a>.</li>
	<li>Select the species (Genome) and Assembly of interest using the dropdown menus.</li>
	<li>Define the Query type using the dropdown menu.</li>
	<li>Paste the sequence of interest into the BLAT Search Genome text box and click Submit.</li>
	<li>Click on the browser link under the ACTIONS heading to navigate to the genomic region of interest.</li>
</ol>
4. Identifying conserved sORFs using PhyloCSF Track Data
<ol>
	<li>Visually scan the genomic area of interest for positively scoring PhyloCSF regions (Figure 1). 
	NOTE: For a detailed explanation of how to visually interpret PhyloCSF scores on the UCSC Genome Browser, see the representative results section below.</li>
	<li>Use the zoom feature to magnify regions of interest to examine sequence characteristics and search for start/stop codons. To zoom in manually, hold the shift key and click and hold the mouse button while dragging along the region of interest. Alternatively, use the zoom in and zoom out buttons at the top of the page to navigate (1.5x, 3x, 10x, or base zoom options are available). 
	NOTE: Before using the zoom in/zoom out buttons, it is necessary to reposition the gene so that the region of interest is in the middle of the screen. To perform this action, click on the image and drag it left or right to move the genomic region horizontally as desired or use the move arrows at the top of the page.</li>
	<li>Zoom in until the nucleotide (base) sequence is visible. 
	NOTE: The nucleotide sequence will appear directly above the +1 Smoothed PhyloCSF score.</li>
	<li>Visually scan the nucleotide sequence near the beginning and end of the positively scoring PhyloCSF regions to identify putative start (ATG) and stop (TGA/TAA/TAG) codons. 
	NOTE: If the gene of interest is on the minus strand of DNA, the start and stop codons will be the reverse complement (i.e., CAT for the start codon and TCA/TTA/CTA for the stop codon).</li>
</ol>
5. Viewing homologous regions in other genomes
<ol>
	<li>Hover the mouse over the View heading at the top of the page and click on the In Other Genomes (Convert) option.</li>
	<li>Define the genome of interest using the dropdown menu below the New Genome heading.</li>
	<li>Select the genomic assembly of interest using the dropdown menu under the New Assembly heading, then click the Submit button.</li>
	<li>Once the browser returns a list of regions in the new assembly with similarity, click on the chromosome position link to navigate to the homologous region of interest. 
	NOTE: The percentage of total bases (nucleotides) and the span that are covered by the region will be defined for each region listed. The higher the percentage of matching bases, the higher the conservation is for the region of interest.</li>
	<li>Follow the same navigational strategies detailed in Section 4 to analyze the sequence.</li>
</ol>
6. Generating multi-species sequence alignments for microproteins of interest
<ol>
	<li>Click on the gene of interest in the GENCODE track on the UCSC Genome Browser (indicated in Figure 1A with a blue box) to navigate to the gene description page.</li>
	<li>Under the Sequence and Links to Tools and Databases heading, click on the link in the table that reads Other Species FASTA.</li>
	<li>Click on the boxes associated with the species of interest to select them. Click on Submit. Copy and paste the sequences appearing at the bottom of the page in FASTA format into a word processing document.</li>
	<li>Open a second browser window and navigate to the Clustal Omega Multiple Sequence Alignment tool52 on the European Bioinformatics Institute (EMBL-EBI) website53,54: <a href="https://www.ebi.ac.uk/Tools/msa/clustalo/">https://www.ebi.ac.uk/Tools/msa/clustalo/</a>.</li>
	<li>Paste the sequence files that are still on the clipboard into the box in STEP 1 that reads sequences in any supported format. Scroll to the bottom of the page and click on Submit. Look below the aligned results (in black font) for symbols that indicate the degree of conservation of each amino acid (symbols are defined in Table 1). 
	NOTE: It may take several minutes to generate the alignment.</li>
	<li>To view the amino acid properties in color, click on the Show Colors link directly above the sequences to color the amino acids according to their properties (defined in Table 2).</li>
	<li>Copy and paste the sequence alignment into a word processing or slideshow program to generate a figure or illustration file (e.g., Figure 2). 
	NOTE: Use a monospaced font for the alignment such as Courier.</li>
	<li>To view other outputs from the Clustal Omega results page, click on the appropriate tabs (i.e., Guide Tree or Phylogenetic Tree).</li>
	<li>Click on the Results Viewers tab for options to view the sequence information using Jalview, a free program that specializes in multiple sequence alignment editing, visualization, and analysis55, or to access direct links to MView and Simple Phylogeny56.</li>
</ol>

The authors declare that they have no competing financial interests.

The protocol presented here provides detailed instructions on how to interrogate genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser48,49,50,51. As detailed above, PhyloCSF is a powerful comparative genomics algorithm that integrates phylogenetic models and codon substitution frequencies to identify evolutionary signatures that a...

The identification of the complete set of coding elements in the genome has been a major goal since the initiation of the Human Genome Project, and remains a central objective toward the understanding of biological systems and the etiology of genetic-based diseases1,2,3,4. Advances in NGS techniques have led to the production of whole genome sequences for an extensive number of organisms, including vertebrates, invertebrates, yeast, and plants5. Additionally, high-throughput transcriptional sequencing methods have further revealed the complexity of the cellular transcriptome, and identified thousands of novel RNA molecules with both protein-coding and noncoding functions6,7. Decoding this vast amount of sequence information is an ongoing process, and challenges remain with comprehensive gene annotation efforts8.
The recent development of translational profiling methods, including ribosome profiling9,10 and poly-ribosome sequencing11, have provided evidence indicating that hundreds of noncanonical translation events map to currently unannotated sORFs throughout the genome, with the potential to generate small proteins called microproteins or micropeptides12,13,14,15,16,17. Microproteins have emerged as a novel class of versatile proteins previously overlooked by standard gene annotation methods due to their small size (&#60;100 amino acids) and lack of classical protein-coding gene characteristics8,12,18,19,20. Microproteins have been described in virtually all organisms, including yeast21,22, flies17,23,24, and mammals25,26,27,28, and have been shown to play critical roles in diverse processes, including development, metabolism, and stress signaling19,20,29,30,31,32,33,34. Thus, it is imperative to continue to mine the genome for additional members of this long-overlooked class of functional small proteins.
Despite the widespread recognition of the biological importance of microproteins, this class of genes remains vastly underrepresented in genome annotations, and their accurate identification continues to be an ongoing challenge that has hindered progress in the field. Various computational tools and experimental methods have recently been developed to overcome the difficulties associated with identifying microprotein-coding sequences (discussed extensively in several comprehensive reviews8,35,36,37). Many recent microprotein identification studies38,39,40,41,42,43,44,45,46,47 have relied heavily on the use of one such algorithm called PhyloCSF48,49, a powerful comparative genomics approach that can be leveraged to distinguish conserved protein-coding regions of the genome from those that are noncoding.
PhyloCSF compares codon substitution frequencies (CSF) using multi-species nucleotide alignments and phylogenetic models to detect evolutionary signatures of protein-coding genes. This empirical model-based approach relies on the premise that proteins are primarily conserved at the amino acid level rather than the nucleotide sequence. Therefore, synonymous codon substitutions, which encode the same amino acid, or codon substitutions to amino acids with conserved properties (i.e., charge, hydrophobicity, polarity) are scored positively, while non-synonymous substitutions, including missense and nonsense substitutions, score negatively. PhyloCSF is trained on whole-genome data and has proven to be effective in scoring short portions of a coding sequence (CDS) in isolation from the full sequence, which is necessary when analyzing microproteins or individual exons of standard protein-coding genes48,49.
Notably, the recent integration of the PhyloCSF track hubs in the University of California Santa Cruz (UCSC) Genome Browser49,50,51 enables investigators of all backgrounds to easily access a user-friendly interface to query genomic regions of interest for protein-coding potential. The protocol outlined below provides detailed instruction on how to load the PhyloCSF track hubs on the UCSC Genome Browser and subsequently interrogate genomic regions of interest to probe for high-confidence protein-coding regions (or the lack thereof). Additionally, in the case where a positive PhyloCSF score is observed, steps are delineated to further analyze microprotein-coding potential and efficiently generate multiple species alignments of the identified amino acid sequences to illustrate cross-species sequence conservation. Lastly, several additional publicly available resources and tools are introduced in the discussion to survey identified microprotein characteristics, including predicted domain structures and insight into putative microprotein function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Website</td>
			<td>Website Address</td>
			<td>Requirements</td>
			<td></td>
		</tr>
		<tr>
			<td>Clustal Omega Multiple Sequence Alignment Tool</td>
			<td><a href="https://www.ebi.ac.uk/Tools/msa/clustalo/">https://www.ebi.ac.uk/Tools/msa/clustalo/</a></td>
			<td>Web browser</td>
			<td>Multiple sequence alignment program for the efficient alignment of FASTA sequences (i.e. for cross-species comparison of identified microproteins)</td>
		</tr>
		<tr>
			<td>COXPRESSdb</td>
			<td><a href="https://coxpresdb.jp">https://coxpresdb.jp</a></td>
			<td>Web browser</td>
			<td>Provides co-regulated gene relationships to estimate gene functions</td>
		</tr>
		<tr>
			<td>EMBL-EBI Bioinformatics Tools FAQs</td>
			<td><a href="https://www.ebi.ac.uk/seqdb/confluence/display/JDSAT/Bioinformatics+Tools+FAQ">https://www.ebi.ac.uk/seqdb/confluence/display/JDSAT/Bioinformatics+Tools+FAQ</a></td>
			<td>Web browser</td>
			<td>Frequently Asked Questions (FAQs) for EMBL-EBI tools. Includes the color coding key for protein sequence alignments</td>
		</tr>
		<tr>
			<td>European Bioinformatics Institute (EMBL-EBI), 
			Tools and Data Resources</td>
			<td><a href="https://www.ebi.ac.uk/services/all">https://www.ebi.ac.uk/services/all</a></td>
			<td>Web browser</td>
			<td>Comprehensive list of freely available websites, tools and data resources</td>
		</tr>
		<tr>
			<td>Expasy - Swiss Bioinformatics Resource Portal</td>
			<td><a href="https://www.expasy.org">https://www.expasy.org</a></td>
			<td>Web browser</td>
			<td>Suite of bioinformatic tools and resources for protein sequence analysis that is maintained by the Swiss Institute of Bioinformatics (SIB)</td>
		</tr>
		<tr>
			<td>National Center for Biotechnology Information (NCBI) 
			Conserved Domain Search</td>
			<td><a href="https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi">https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi</a></td>
			<td>Web browser</td>
			<td>Search tool to identify conserved domains within protein or coding nucleotide sequences</td>
		</tr>
		<tr>
			<td>Pfam 35</td>
			<td><a href="http://pfam.xfam.org">http://pfam.xfam.org</a></td>
			<td>Web browser</td>
			<td>Protein family (Pfam) database, provides alignments and classification of protein families and domains</td>
		</tr>
		<tr>
			<td>PhyloCSF Track Hub Description</td>
			<td><a href="https://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=1267045267_TEc99h2oW5QedaCd4ir8aZ65ryaD&#38;db=mm10&#38;c=chr2&#38;g=hub_109801_PhyloCSF_smooth">
https://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=1267045267_TEc99h2oW5Q edaCd4ir8aZ65ryaD&#38;db=mm10 &#38;c=chr2&#38;g=hub_109801_ PhyloCSF_smooth</a></td>
			<td>Web browser</td>
			<td>Detailed description of the Smoothed PhyloCSF tracks and PhyloCSF Track Hub &#160; &#160;&#160; &#160;&#160; &#160;&#160; &#160;&#160; &#160;</td>
		</tr>
		<tr>
			<td>SignalP 6.0</td>
			<td><a href="https://services.healthtech.dtu.dk/service.php?SignalP-6.0">https://services.healthtech.dtu.dk/service.php?SignalP-6.0</a></td>
			<td>Web browser</td>
			<td>Predicts the presence of signal peptides and the location of their cleavage sites</td>
		</tr>
		<tr>
			<td>TMHMM - 2.0</td>
			<td><a href="https://services.healthtech.dtu.dk/service.php?TMHMM-2.0">https://services.healthtech.dtu.dk/service.php?TMHMM-2.0</a></td>
			<td>Web browser</td>
			<td>Prediction of transmembrane helices in proteins</td>
		</tr>
		<tr>
			<td>UCSC Genome Browser BLAT Search</td>
			<td><a href="https://genome.ucsc.edu/cgi-bin/hgBlat">https://genome.ucsc.edu/cgi-bin/hgBlat</a></td>
			<td>Web browser</td>
			<td>Tool used to find genomic regions using DNA or protein sequence information</td>
		</tr>
		<tr>
			<td>UCSC Genome Browser Gateway</td>
			<td><a href="https://genome.ucsc.edu/cgi-bin/hgGateway">https://genome.ucsc.edu/cgi-bin/hgGateway</a></td>
			<td>Web browser</td>
			<td>Direct link to the UCSC Genome Browser Gateway</td>
		</tr>
		<tr>
			<td>UCSC Genome Browser Home</td>
			<td><a href="https://genome.ucsc.edu/">https://genome.ucsc.edu/</a></td>
			<td>Web browser</td>
			<td>Home website for the UCSC Genome Browser</td>
		</tr>
		<tr>
			<td>UCSC Genome Browser Track Data Hubs</td>
			<td><a href="https://genome.ucsc.edu/cgi-bin/hgHubConnect#publicHubs">https://genome.ucsc.edu/cgi-bin/hgHubConnect#publicHubs</a></td>
			<td>Web browser</td>
			<td>Direct link to Track Data Hubs/Public Hubs database to search for and load the PhyloCSF Tracks</td>
		</tr>
		<tr>
			<td>UCSC Genome Browser User Guide</td>
			<td><a href="https://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html">https://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html</a></td>
			<td>Web browser</td>
			<td>Comprehensive user guide detailing how to navigate the UCSC Genome Browser</td>
		</tr>
		<tr>
			<td>WoLF PSORT</td>
			<td><a href="https://wolfpsort.hgc.jp">https://wolfpsort.hgc.jp</a></td>
			<td>Web browser</td>
			<td>Protein subcellular localization prediction tool</td>
		</tr>
	</tbody>
</table>

an integrated approach for microprotein identification and sequence analysis

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model organisms. However, despite this wealth of sequence information, comprehensive gene annotation efforts have proven challenging, especially for small proteins. Notably, conventional protein annotation methods were designed to intentionally exclude putative proteins encoded by short open reading frames (sORFs) less than 300 nucleotides in length to filter out the exponentially higher number of spurious noncoding sORFs throughout the genome. As a result, hundreds of functional small proteins called microproteins (&#60;100 amino acids in length) have been incorrectly classified as noncoding RNAs or overlooked entirely.
Here we provide a detailed protocol to leverage free, publicly available bioinformatic tools to query genomic regions for microprotein-coding potential based on evolutionary conservation. Specifically, we provide step-by-step instructions on how to examine sequence conservation and coding potential using Phylogenetic Codon Substitution Frequencies (PhyloCSF) on the user-friendly University of California Santa Cruz (UCSC) Genome Browser. Additionally, we detail steps to efficiently generate multiple species alignments of identified microprotein sequences to visualize amino acid sequence conservation and recommend resources to analyze microprotein characteristics, including predicted domain structures. These powerful tools can be used to help identify putative microprotein-coding sequences in noncanonical genomic regions or to rule out the presence of a conserved coding sequence with translational potential in a noncoding transcript of interest.

Here we will use the validated microprotein mitoregulin (Mtln) as an example to demonstrate how a conserved sORF will generate a positive PhyloCSF score that can be easily visualized and analyzed on the UCSC Genome Browser. Mitoregulin was previously annotated as a noncoding RNA (formerly human gene ID LINC00116 and mouse gene ID 1500011K16Rik). Comparative genomics and sequence conservation analysis methods played a critical role in its initial discovery40,<sup class=...

Watch this Scientific Journal Video about An Integrated Approach for Microprotein Identification and Sequence Analysis at JoVE.com

An Integrated Approach for Microprotein Identification and Sequence Analysis

The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model ...

an-integrated-approach-for-microprotein-identification-sequence

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3.2K Views.  Cincinnati Children's Hospital Medical Center. The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.

Video: An Integrated Approach for Microprotein Identification and Sequence Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR.
Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well.
The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors 9. In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...