Proteins that are attached to DNA

[Dory]

I know only of histones, and their role in creating nucleosome...but what are some other proteins that are in the chromosome, and what are their functions?

[GenesForLife]

Condensisns

http://en.wikipedia.org/wiki/Condensin

Condensins are large protein complexes that play a central role in chromosome assembly and segregation in eukaryotic cells.

In vertebrate cells, at least two different types of condensin complexes, condensin I and condensin II, are known. The two complexes share the same pair of core subunits, SMC2 and SMC4, both belonging to a large family of chromosomal ATPases, known as SMC proteins. SMC stands for Structural Maintenance of Chromosomes. Each of the complexes contains a distinct set of non-SMC regulatory subunits.

In human tissue culture cells, the two condensin complexes are regulated differently during the cell cycle. Condensin II is present within the cell nucleus during interphase and is involved in an early stage of chromosome condensation within the prophase nucleus. On the other hand, condensin I is present in the cytoplasm during interphase, and gains access to chromosomes only after the nuclear envelope breaks down at the end of prophase. During prometaphase and metaphase, both condensin I and condensin II contribute to the assembly of condensed chromosomes, in which two sister chromatids are fully resolved. The two complexes apparently stay associated with chromosomes after the sister chromatids separate from each other in anaphase. At least one of the subunits of condensin I is known to be a direct target of a cyclin-dependent kinase (Cdk).

The structure and function of condensin I are conserved from yeast to humans, but yeast has no condensin II. In the nematode, condensin II appears to play a major role in chromosome assembly and segregation, whereas a condensin I-related complex participates in chromosome-wide gene regulation, i.e., dosage compensation. Even in bacterial cells, ancestral forms of condensins regulate the organization and segregation of their chromosomes (nucleoids).

A distinct yet structurally related protein complex, cohesin, is involved in sister chromatid cohesion. They contain a different pair of SMC subunits, SMC1 and SMC3, as their core ATPase subunits.

[Dory]

Awesome, thanks Genes. You're just a bundle of help lately.

[GenesForLife]

One more , this one actually follows pretty obviously from Mitosis, centromeric DNA has to be associated with kinetochore proteins which can in turn bind to microtubules that make up spindle fibers.

Here is a particularly interesting descriptive manuscript on kinetochores and DNA.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2112771/

The kinetochore is the protein-DNA complex at eukaryotic centromeres that functions as the attachment site for spindle microtubules. In budding yeast, the centromere spans 120 bp, there is a single microtubule per kinetochore, and the entire spindle is composed of 16 kinetochore microtubules plus four interpolar microtubules from each pole. There are >65 different proteins at the kinetochore, organized in at least six core multimeric complexes [1]. A spindle checkpoint network monitors the state of attachment and tension between the microtubule and chromosome. We present a model for the path of DNA in the kinetochore.
Replicated sister centromeres become maximally separated by 600-800 nm in metaphase [2]. Separation progressively decreases along chromosome arms such that sister chromatids are tightly juxtaposed at ∼10 kb from the centromere [2]. The molecular glue linking sister chromatids, cohesin, is recruited to a 20-50 kb region surrounding the centromere at 3- to 5-fold higher levels than centromere-distal locations [3]. A major paradox is the accumulation of cohesin at regions of separated sister DNA strands. A second problem is the nature of the mechanical linkage coupling DNA to a dynamic microtubule plus-end. This linkage must resist detachment by mitotic forces while sliding along the polymerizing and depolymerizing microtubule lattice.
We propose that pericentric chromatin is held together via intramolecular cohesion (Figure 1), similar to a foldback structure proposed for the fission yeast centromere [4]. In contrast to fission yeast, the budding yeast core centromere (120 bp DNA wrapped around a specialized nucleosome containing two molecules of the centromere-specific histone H3 variant, Cse4) and flanking chromatin may adopt a cruciform configuration in metaphase.

The reference for the bolded bit is stuck behind a paywall though.

A free compilation of research articles from Pubmed contains 305 papers, and can be found at http://www.ncbi.nlm.nih.gov/pubmed

As far as being helpful is concerned, I'd say it's my pleasure.

[Dory]

65 different proteins in the kinetochore alone!? Holy smokes!

I was initially thinking you were referring to dyenin and kinesin ....but damn, I guess with 65 proteins life is very, very, very complex even at the kinetochoric level.

And if the kinetochore alone has 65 proteins, I can just imagine there are plenty more to the entire chromosome than histones and condensins.

[GenesForLife]

These chaps intend to map 125 of them.

http://www.modencode.org/Karpen.shtml

[Dory]

Yea, in a damn fruit fly! Why not in a human, I wonder?

I presume that numbers and types of chromosomes very much vary across species, though I'm sure there are basic similiarities too.

[GenesForLife]

Yea, in a damn fruit fly! Why not in a human, I wonder?

I presume that numbers and types of chromosomes very much vary across species, though I'm sure there are basic similiarities too.

One reason for D.melanogaster being used , I'd imagine is the presence of extremely large salivary gland chromosomes, high resolution results can be obtained for fluorescent tagging experiments, either by FISH or by antibody tagging, while the numbers may vary across species, Dory, I'd imagine the basic nonhistone protein set to be conserved across the domain Eukarya, more or less.

Evolution makes model organisms possible, ranging from Drosophila to primates to good ol' Nobel winning Caenorhabditis elegans

[Dory]

Yea, in a damn fruit fly! Why not in a human, I wonder?

I presume that numbers and types of chromosomes very much vary across species, though I'm sure there are basic similiarities too.

One reason for D.melanogaster being used , I'd imagine is the presence of extremely large salivary gland chromosomes, high resolution results can be obtained for fluorescent tagging experiments, either by FISH or by antibody tagging, while the numbers may vary across species, Dory, I'd imagine the basic nonhistone protein set to be conserved across the domain Eukarya, more or less.

Evolution makes model organisms possible, ranging from Drosophila to primates to good ol' Nobel winning Caenorhabditis elegans

I see... can't argue with that.

[GenesForLife]

If I could get a list of those kinteochore proteins, for I could run a database search and BLAST it, and see if there are any conserved domains, a built in distance tree, if it indicates a concestor before plants and animals, would indicate that those proteins, or the domain at least, originated before the metazoa-metaphyta split in Eukarya.

[Dory]

If I could get a list of those kinteochore proteins, for I could run a database search and BLAST it, and see if there are any conserved domains, a built in distance tree, if it indicates a concestor before plants and animals, would indicate that those proteins, or the domain at least, originated before the metazoa-metaphyta split in Eukarya.

I'd imagine BLAST is one of those bioinformatics tools and that the metazoa-metaphyta split has something to do with early protists? It's a bit of a fancy talk for me truth be told.

[GenesForLife]

If I could get a list of those kinteochore proteins, for I could run a database search and BLAST it, and see if there are any conserved domains, a built in distance tree, if it indicates a concestor before plants and animals, would indicate that those proteins, or the domain at least, originated before the metazoa-metaphyta split in Eukarya.

I'd imagine BLAST is one of those bioinformatics tools and that the metazoa-metaphyta split has something to do with early protists? It's a bit of a fancy talk for me truth be told.

yeah, you've got both right, if you're interested I can help you learn sequence analysis, it is easy and I learned it in two weeks.

[Dory]

If I could get a list of those kinteochore proteins, for I could run a database search and BLAST it, and see if there are any conserved domains, a built in distance tree, if it indicates a concestor before plants and animals, would indicate that those proteins, or the domain at least, originated before the metazoa-metaphyta split in Eukarya.

I'd imagine BLAST is one of those bioinformatics tools and that the metazoa-metaphyta split has something to do with early protists? It's a bit of a fancy talk for me truth be told.

yeah, you've got both right, if you're interested I can help you learn sequence analysis, it is easy and I learned it in two weeks.

Awesome. I'm actually really enjoying our back-and-forth here.

And are you kidding me? Of course I'm game. I love this stuff. You'd really help me learn it? I'm not entirely sure what I'm in for, but I'm enticed. If you're serious I'll happily send you my contact details-- I'm online all day, and I'm studying all the time -- when I'm not sleeping. So, as far as time-management, no worries. Either way, you're a bundle of help...I'm glad I'm sticking to Ratz with my science questions, it's providing the best results contrary to my belief...compare to Naked Scientists and other forums that I expected better.

[GenesForLife]

Yes, I'm serious. To get things started off, I'll post a little text tutorial I prepared to explain BLAST to someone else, it is by no means exhaustive, and is meant to be a very general instruction set.

1) Basically sequences either come in nucleotides or in amino acids (which is represented by a single letter code called the FASTA format)

2) You need to go to the NCBI website.

3) There are two possible approaches here, if you are looking for a sequence to use as a standard, say for example Hox genes, type the name in the search box and choose either nucleotide or protein in the drop down next to the search bar.

4) This will bring up a list of database entries, choose whatever you are interested in using as a standard, and to the right side of the page there is a link to run BLAST.

5) The BLAST tool page offers you the option of limiting your work to specific taxonomic divisions/groups , just type in a few letters and select what you want.

6) Run BLAST.


The other approach involves going to BLAST directly, then typing in the sequence you're looking for in the Query sequence box, and proceeding, the algorithm will take some time and eventually give you a set of results, there is an option to view the phylogenetic distance tree of the processed results based on similarity searches at the bottom of the results page.

[GenesForLife]

example...

ndm-1 protein , the one that has been causing a scare with antibiotic resistance.

Using the search terms ndm-1 and setting the search filter to nucleotide, I retrieved this database page.

http://www.ncbi.nlm.nih.gov/nuccore/300422615

LDMPGFGAVASNGLIVRDGGRVLVVDTAWTDDQTAQILNWIKQE
INLPVALAVVTHAHQDKMGGMDALHAAGIATYANALSNQLAPQEGMVAAQHSLTFAAN
GWVEPATAPNFGPLKVFYPGPGHTSDNITVGIDGTDIAFGGCLIKDSKAKSLGNLG

is the amino acid sequence, this is in the single letter code, look it up if you want to convert this to amino acid names, but it will not be required usually, and is produced by this nucleotide transcript.

1 tctcgacatg ccgggtttcg gggcagtcgc ttccaacggt ttgatcgtca gggatggcgg
61 ccgcgtgctg gtggtcgata ccgcctggac cgatgaccag accgcccaga tcctcaactg
121 gatcaagcag gagatcaacc tgccggtcgc gctggcggtg gtgactcacg cgcatcagga
181 caagatgggc ggtatggacg cgctgcatgc ggcggggatt gcgacttatg ccaatgcgtt
241 gtcgaaccag cttgccccgc aagaggggat ggttgcggcg caacacagcc tgactttcgc
301 cgccaatggc tgggtcgaac cagcaaccgc gcccaacttt ggcccgctca aggtatttta
361 ccccggcccc ggccacacca gtgacaatat caccgttggg atcgacggca ccgacatcgc
421 ttttggtggc tgcctgatca aggacagcaa ggccaagtcg ctcggcaatc tcggt

To the task pane on the right side of the database entry, you can see, under the section "Analyze this sequence" , a link to "Run BLAST"

Click it.

Because I'm looking for a wide range of related sequences in this case, I chose nBLAST, if you're looking for exceptionally conserved sequences, you can choose megablast in the options form, you'll have a better idea once you play around with the different options available.

Click BLAST.

This is the results page http://blast.ncbi.nlm.nih.gov/Blast.cgi ... RY_INDEX=0

The Distance tree I spoke about is present here http://blast.ncbi.nlm.nih.gov/blast/tre ... xh=&ns=100

It looks like the sequence that was analyzed either underwent Horizontal Gene Transfer between Klebsiella and E.coli or diverged from an ancestral sequence present in a concestor (which can be confirmed by checking out their taxonomic relationship, which the tree indicates is close as both are enterobacteria.

That is just one example of BLAST being used.

The other option, the PCR Primer design thing on the database entry page is good for designing probes so that you can see, using PCR and Electrophoresis, if that particular sequence is present in DNA from bacterial isolates.