journal 2 post quiz

question

what is the open reading frame (ORF) seq for human SOX7 (=part of dna seq that does not have a stop codon so that rna code is continuously read) + compare to mouse sox7 to see if conserved

method

pcr

result

orf seq has hmg box. 87.4% similarity btn human and mouse sox7. 100% overlap for hmg box

control

mouse sox7 establish seq to compare human seq to

conclude

high fx similarity btn human and mouse. fx of mouse sox7 protein=fx of human sox7 protein. conserved

classify

establishing, expression****

question

where are sox7 genes in mouse and human chromosomes

method

fish mapping

conditions

human bac and mouse pac

result

green signals: mouse sox7 at chromosome 14 band D. human sox7 at chromosome 8 p22 which is similar seq

control

mouse pac establish baseline localization to compare human loc to

conclude

locs are similar. fx of mouse sox7 protein=fx of human. loc of sox7 gene on human is where syndromes→ sox7 associated with syndromes

classify

establishing, other=flourescene ver of southern blot

question

how is sox7 mrna expressed in mice

method

northern blot

conditions

fetal and adult tissues of mice + probe

result

sox7 expressed in all fetal and adult mouse tissue at 4kb with higher exp in heart and lungs for both embryonic and adult

control

28s is rrna that is always exp at same levels → shows equal loading + ethidium bromide staining to show if equal amt of rna in each lane

conclude

sox7 plays a role in all tissues (contrary to prev study) but is particularly significnat in dev of heart and lungs

classify

hypothesis/establishing, expression

question

where is sox7 mrna exp in humans

method

northern blot

conditions

fetal and adult tissue

result

sox7 exp in all fetal and adult human tissue at 5kb. additional 2 bands at 2.5 kb for adult colon and fetal lung

control

b actin for equal loading to verify rna is present

conclude

sox7 plays role in all tissues. bands at 2.5 kb can be splice variants or unspliced messengers.

classify

hypothesis/establishing, expression

question

where sox7 is exp as mouse embryo devlop over time 8 dpc

method

whole mount in situ hybridisations

conditions

mouse embryo at 8 to 11.5 and 17.5 dpc

result

at 8 dpc, sox7 is exp in somites (black) and brain (white)

control

sense probe = negative control that would show no staining. rna probe is antisense to sox7 mrna. the sense probe wouldn’t bind to sox7 mrna.

conclude

sox7 is exp in the regions that need to be developed. there is early exp and restricted head dev.

classify

hypothesis, exp

question

where sox7 is exp in a developing mouse embryo 9.5 dpc

method

in situ hybridisations

conditions

mouse embryo at 8 to 11.5 and 17.5 dpc

result

at 9.5 dpc, sox7 is exp thru vascular system in intersomitic vessels. sox7 helps create the endothelial cells which are the cells inside skin of vessels. exp of sox7 makes the endothelial cell the cell that it is

control

sense probe

conclude

sox7 plays a role in vascular development

classify

hypothesis, exp

question

where is sox7 exp in developing mouse embryo 11.5 dpc

method

in situ hybridisations

conditions

mouse embryo at 8 to 11.5 and 17.5 dpc

result

at 11.5 dpc, sox7 is mainly exp in intersomitic vessles

control

sense probe

conclude

exp of sox7 plays a role in development of mouse embryo

classify

hypothesis, exp

question

where is sox7 exp in developed embryo 17.5

method

in situ hybridisations

conditions

mouse embryo at 17.5 dpc vs 8-11.5

result

at 17.5 dpc, sox7 is seen in brain, cochlea, tongue, cartilage, lung, liver, vertebrae. starts being restricted

control

sense probe=negative control & positive control=nuclear fast red staining=counterstain that stains all structures + probe show where mrna is exp

conclude

after threshold of 17.5 dpc, exp becomes restricted to certain site

classify

hypothesis, exp

question

where is sox7 exp in human and if conserved btn mouse and human

conditions

8 week human embryo at carnegie stage 19 vs 17.5 dpc mouse

result

8 week embryo, sox7 is in brain tongue heart liver lung vertebrae in pattern similar to mouse

control

negative control=sense probe, positive control=nuclear fast red stain=counterstain that stains everything + probe that shows where mrna exp

conclude

sox7 plays a role in development AND differention and exp gets restricted like mice → sox7 is conserved btn mouse and embryo. those organs are where epithelial mesencymal transition happen, so sox7 is involved in emt

classify

hypothesis, exp

question

how is sox18 expressed compared to sox7

method

northern blot

conditions

sox18 probe + embryonic vs adult

result

exp of sox18 had similar pattern to sox7 for both embryo and adult: exp in all tissues but particularly high in heart and lung

control

28s is rrna that is always exp at same levels → shows equal loading + ethidium bromide staining to show if equal amt of rna in each lane

conclude

sox7 is comparable to sox18 → fxs overlap → genes come from same ancestor

classify

hypothesis/establishing, exp

question

where is sox18 exp compared to sox7. do they colocalize in intersomitic vessels of 11.5 dpc mouse

method

fish

conditions

sox7 antisense prob, sox18 antisense probe

result

mrna exp had a lot of overlap = yellow

control

sense probe

conclude

sox7=sox18 in seq and exp → fxs overlap → genes come from same ancestor

classify

hypothesis, subcell loc

question

is sox7 a transcription activator or repressor and what section is responsible for activation

method

relative luciferase assay

conditions

full length sox7/sox7 deletion mutants; soxluc reporter plamsid that has motif/sacluc vector with no motif

result

FL sox7 had high soxluc activity → sox7 binds to the dna binding site in reporter plasmid → sox7 is a transcription activator; mutants were not activators

control

mutated sox7 with the c terminus cut off → baseline fx of sox7 with just hmg domain. sacluc → baseline activity of sox7. pglomyc=negative control=has nothing in it so won’t have any activity

conclude

transcription activation is loc in cterminus of sox7.

class

hypothesis, functional (does that seq work and what is it doing)

question

does sox7 affect wnt singaling pathway

method

relative luciferase assay

conditions

everything on x axis, bcatenins33 not regular bcatenin bc its the active stable ver + topflash=reporter plasmid that has bindnig sites, fopflash=plasmid with mutated bcatenin

result

flsox7+bcatenin < bcatenin alone → flsox7 represses luciferase reporter’s ability to activate b catenin

control

empty pglomyc = wild type seq with no b catenin → no bcatenin=nothing to activate. fopflash=mutated so nothing activates it

conclude

yes, sox7 impacts wnt singalling pathway. reg genes of wnt pathway get mutated in cancer → sox7 is tumor suppresor gene

class

hypothesis, functional