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Chromosomes and DNA Packaging--lecture

发布时间:2013-12-04 14:02:49  





?Human genome (in diploid cells) = 6 x 109bp6 x 109bp X 0.34 nm/bp = 2.04 x 109nm = 2 m/cellVery thin (2.0 nm), extremely fragileDiameter of nucleus = 5-10 mm ?DNA must be packaged to protect it, but must still be accessible to allow gene expression and cellular responsiveness

?Single DNA Molecule and associated proteins?Karyotype

?Chromatin vs. Chromosomes

?Main packaging proteins

?5 classes: H1, H2A, H2B, H3, H4. ?

Rich in Lysine and Arginine

?NOTE: if histones from different species are added to any eukaryotic DNA sample, chromatin is reconstituted. Implication?

?Very highly conserved in eukaryotes in both




?Must allow gene activity

?Fairly uniform, some variability

?Why variable if only function is packaging?

?How can cells introduce changes in protein structure and thus protein function?


–Post transcriptional modifications—ex alternate splicing

–Post translational modifications


??Methylation–NOTE: These processes are dynamic. They give the cell another means to regulate gene expression

DNA is complexed with proteins and organized as chromatin in

the interphase nucleus:

Fig. 9

structure from naked DNA to chromatin to fully condensed chromosomes...

10 nm filament; nucleosomes

deoxyribonuclease I (DNase I) digestionSeparate DNA from protein





2H4DNAoctamernucleosome protect 146 nt from DNase I attack.

10 nm Fiber

?A string of nucleosomes is seen under EM as a 10 nm


30 nm Fiber

?30 nm fiber is coil of nucleosomes with


Fig. 9

structure from naked DNA to chromatin to fully condensed chromosomes...

Acid extraction removes histones from meta-phase chromosomes, leaving nonhistones...

Low power high powerDNAloops


Major non-histone proteins = topoisomerases!

Different forms of chromatin show differential gene activity.



?In sperm heads, DNA must be further compressed?Protamines

?~2/3rdbasic amino acids, (mostly Arg)

?Greater degree of compaction associated with:

–Fit in DNA

–Salt bridges between Arg residues in both the contact helix and adjacent helices

?Gene expression in a dynamic environment ?DNA associated non-histones:

–Palindromes?-helicesSymmetrical dimersInteraction between R-groups and specific basesRole of basic amino acids

?How will this affect His’s ability to participate in

weak interactions?

Euchromatin (E) vsHeterochromatin (H)Fig. 11


Being more condensed (tightly packed), heterochromatin is resistant to DNase digestion.


Transcriptionally active DNA (i.e., an

active gene) is in euchromatin.Hpreferentially digest active genes in a cell...

An Experiment:partially digested

chromatin; extract

make 2

of these...

Increasing time of digestionFig. 12

?Radiolabel, denature cloned globin and other geneDNA to use as *probe.

?Allow *probeDNA to hybridizeto complementarysequences on filters… expose, develop autoradio-graph...

Increasing time of digestion

globin gene probe

ovalbumin gene probeDNA from digested RBC chromatin

Fig. 13


?Compared to globin gene DNA, ovalbumin geneDNA in RBC chromatin resists DNase digestion. ?Globin gene DNA in RBC chromatin must bemore accessible to DNase (less condensed,more euchromatic) than ovalbumin DNA.


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