LAN connections use on-site hardware servers to funnel users through ethernet links to organizational internet host computers (or to an ISP). All internet connections require a special communication software called TCP/IP (Transmission Control Protocol/Internet Protocol).

Aside from the use of e-mail and on-line journal publications, the primary research-related activities are;

1) DNA sequence analysis using sequence alignment programs,

2) Searching integrated databases to locate biological resources and reagents, and

3) Interactive communication with specialized biological sciences user groups.

DNA sequence analysis programs and databases

One of the most convenient methods to search public domain databases is to use an internet connection to search GenBank with the Basic Local Alignment Search Tool (BLAST). GenBank is maintained by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine which is located on the NIH campus in Bethesda, Maryland. Information about BLAST is readily avalaible through the NCBI web site.

GenBank is a member of the International Nucleotide Sequence Database Collaboration, an organization that includes the European Molecular Biology Laboratory (EMBL) and the DNA DataBank of Japan (DDBJ). The GenBank, EMBL and DDBJ databases freely exchange DNA sequence files on a daily basis to maintain a comprehensive set of all known sequences.

You should get a result that looks something like this:

The results of BLAST search queries are used to design laboratory experiments that test the biological relevance of DNA and protein sequence homologies. For example, a BLAST search using coding sequences from the hypothetical mouse AMG gene could be done to determine if there are any homologous or orthologous (evolutionarily conserved) gene sequences in GenBank.


Three possible outcomes are illustrated;

(a) AMG identity with a previously cloned mouse cDNA.

(b) Limited, but highly significant homology, with several mouse and human genes.

(c) Yeast gene is found that shares the same region of homology as the mouse and human homologues.

Integrated biological resource databases
One of the most exciting molecular genetic applications of the WWW has been the ability to exploit hypertext file linking functions using strategies that create internet entry points for integrated resource databases. The basic idea of these linked web sites has been to enable researchers to place DNA sequence information into a proper biological context.

One example of an integrated database resource is the GenomeNet project in Japan, which is operated jointly by Kyoto University and the University of Tokyo. GenomeNet is a WWW-accessible network of computational services and databases that were initially developed as a research tool for molecular and cell biological studies in Japan.

Try out GenomeNet by researching enzymes involved in Metabolic Pathways.

Bionet newsgroups for molecular genetic research
So far we have only described ways that molecular genetic researchers use the internet to obtain information, but it is also an important means of communication between individuals (e-mail), and between groups of users that have a common interest (newsgroups or forums). Internet newsgroups function like electronic bulletin boards and have been around since the inception of the internet network.

One of my favorite protein structures is the glucocorticoid receptor bound to DNA. You can also investigate glucocorticoid receptor structure and function using a steroid receptor integrated database put together by researchers at George Washington University.

What do you conclude about the possible function of the "AMG Test Protein" based on the GenBank query results?

What is the primary difference between the "nr" and "dbest" databases? What is the difference between a "blastn" search and a "tblastx" search?

Is the yeast genome sequence included in the "nr" database, or do you have to search the "yeast" database specifically to find ortholgous genes?


Which integrated Internet database is used to query protein structure information? Where would you go on the Internet to find text abstracts from published research articles?

What is meant by the team "database mining" in the context of applied molecular genetics?

Functional Genomics and Proteomics using Mass Spectrometry

The availability of the entire DNA sequence of an organism presents many opportunities for gene analysis. The term "functional genomics" refers to the study of genome expression, i.e., assigning function to every gene product encoded by the genome. Since RNA represents an intermediate "messenger" in genomic expression, it has limited usefulness - one of which is high throughput "profiling" of genomic expression by microarrays. However, it is really the proteins in a cell that carry out the business of biochemical life. Indeed, gene expression in the functional sense, really refers to protein synthesis and biological control.

The study of an organism's "proteome" is made possible by a new approach to biochemical analysis called "proteomics." Proteomics is the large scale analysis of gene functions at the protein level. The major tool in proteomic research is Mass Spectrometry. The birth of proteomics can be traced back to at least the1970s when Pat O'Farrell developed the use of two-dimensional protein gels to analyze thousands of proteins at a time in cell extracts. Modern day proteomics combines this "proteome" approach with sensitive instrumentation in the form of mass spectrometry. The key to proteomics is the use of powerful computer algorithms that search genomic databases to match protein mass data with theoretical protein masses. This strategy of using genomic databases as a tool to understand gene function is a major component of bioinformatics.

Mass spectrometry measures the mass of particles by several different methods based on determing the mass to charge ratio. Mass spectrometery requires charged gaseous molecules for analysis. Up until the 1980s mass spectrometry had few applications in the life sciences because native proteins and peptides are large polar molecules that are not easily transferred to gas phase and ionized. Two techniques developed in the late 1980s changed all of this by providing a way to ionize proteins that could then be analyzed by mass spectrometry.


The first method was developed by Karas and Hillenkamp and is called MALDI for matrix-assisted laser desorption ionization. MALDI is based on coprecipitating peptides with matrix material that is then dried onto a metal surface and irradiated with nanosecond laser pulses to ionize the peptides and create a gas phase. The precise mechanism of MALDI-based ionization is not known. MALDI is often coupled to another technique called time of flight (TOF) analysis which is how the charge to mass ratio is determined (MALDI-TOF).

The second method is called electrospray mass spectrometry or electrospray ionization (ESI) which was first described in 1989 by Fenn and coworkers. ESI utilizes a mixture of peptides in acid (MeOH or acetonitrile) that is pumped through a hypodermic needle at high voltage to electrostatically disperse small droplets which evaporate and impart charge onto the peptide. This gaseous material contains charged peptides that are suitable for mass spectrometry analysis.

Mass spectrometry has three primary roles in proteomic applications

- Quality control for protein production in biotechnololgy

- Protein identification in biochemical research

- Analysis of post-translational modification of proteins



The use of analytical tools such as mass spectrometry to investigate proteome dynamics has benefited greatly by the availability of the complete sequence of organismal genomes. The reason for this is that bioinformatics can then predict the entire set of proteins (or at least hypothetical proteins) that an organism is capable of expressing, and this set is used to interpret mass spectrometry data.

Two main applications of proteomics to functional genomics are:

Expression Proteomics - identifying proteins that are up- or down-regulated (2D gels).

Functional Proteomics - identifying proteins that are part of large complexes (Co-IP expts.)

Let's look at one of the most powerful applications of mass spectrometry in proteomics, the identification of co-immunopreciptated proteins using epitope-tagged "bait" proteins, a strategy that supersedes the yeast two-hybrid approach. The ability to identify proteins of very low abundance from protein gel slices (5-50ng of a 50 kDa protein or 0.1 - 1 pmol) makes the co-immunoprecipitation method more reliable for in vivo interactions since it does not depend on fusion protein functions in yeast cells.


A strategy for characterizing interacting proteins by co-immunoprecipitation (see Protein Interactions):

Cleavage of proteins into peptides using trypsin is the first step in protein identification by mass spectrometry. Trypsin cleaves polypeptides on the carboxy terminal side of Lysine (K) or Arginine (R) residues. Therefore, analysis of peptides by mass spectrometry includes the tentative assignment of the carboxyl terminal residue as a lysine or arginine with a known mass.

In tandem mass spectrometry (MS/MS), a mixture of peptides is separated out into individual peptides that are energized and susceptible to breakage at peptide bonds. The mass of the resulting peptide fragments are then determined and the protein sequence is deduced by calculating the masses of fragments differing by one amino acid. Tryptic fragments derived from the C-terminal end contain a Lys or Arg residue and ar called the "y" ions, fragments with a common N-terminal are called the "b" ions. Some amino acids cannot be resolved because their masses are very similar, for example, the mass of isoleucine is exactly the same as the mass of leucine.

Importantly, protein sequence determination by mass spectrometry requires comparison of the determined masses to predicted peptides in genomic databases. Three different ways to identify proteins by mass spectrometry using Genomic Database queries: