What do scaffold proteins really do (on-going)

What do sca?old proteins really do?

(on-going)

Mu?y Calder?Jane Hillston?

July24,2006

Abstract

The role of sca?old proteins within signal transduction pathways is an area of current active investigation by biochemists.Much of their

e?ort is presently directed towards identifying which of the many proteins

implicated in such pathways,play this role.Moreover the full biological

signi?cance of the role is yet to be fully understood.It seems that generic

models of the dynamic properties of sca?old proteins and their in?uence

on the signalling can be helpful in interpreting experimental data and

developing understanding.We have been developing some PEPA models

of the role of sca?old proteins.In this paper we report the results of our

preliminary experiments.

1Introduction

In this paper we present some preliminary work on using PEPA to model sca?old proteins,and investigating their role within the dynamic behaviour of signal transduction pathways within cells.We do not look at any particular pathway, but rather develop generic models which seek to illustrate how the sca?old proteins interact with other proteins in the pathway,and the impact that this has on the signalling dynamics.

This work is just in its infancy and we present here two simple models.The ?rst represents only the formation of the complex on the sca?old and how this is a?ected by the concentrations of the sca?old and the reagents,and by the ratio between the binding and release rates.This model is analysed using both Markovian and continuous state space semantics.The second model considers the competitive formation of protein complexes in the cytosol,assuming that once proteins have bound in the cytosol they are no longer able to engage with the sca?old.This model is analysed only using the continuous state space semantics.For both models careful consideration of the kinetics is needed,as will be explained.

The rest of the paper is structured as follows.In Section2we present a brief introduction to sca?old proteins and their role in cell signalling.Section3 presents the?rst PEPA model,considering the binding of reagents to sca?old,?Department of Computing,University of Glasgow,Scotland. Email:muffy@https://www.wendangku.net/doc/234703985.html,

?School of Informatics,University of Edinburgh,Scotland.Email:jeh@https://www.wendangku.net/doc/234703985.html,

1

Figure1:Schematic view of a sca?old protein

and the numerical experiments upon it.Section4presents the elaborated model which takes into account the formation of complexes in the cytosol.Finally,in Section5we summarise our results and discuss some directions for future work. 2Sca?old proteins

In signal transduction pathways within a cell,a series of biochemical reactions serve to pass a message from the cell membrane to the nucleus.The message is prompted by the arrival of a signalling molecule at the membrane of the cell, and the result of the signal can be a profound e?ect on the behaviour of the cell,for example triggering cell replication,di?erentiation or apoptosis.The message is passed as peaks of concentration of proteins along the pathway and sophisticated structures in the pathway can mean that the message is ampli?ed or attenuated as it passes from membrane to nucleus.

Considerable wet lab e?ort is being applying to identifying the proteins which constitute di?erent signalling pathways.This experimental work has identi?ed some proteins which appear to play an organisational rather than a signalling role.These proteins,termed sca?old proteins,serve as additional infrastructure within the cell rather than actually carrying the message themselves[1].The exact purpose of this infrastructure is not yet clear although it is possible that by increasing local concentrations the sca?olds enhance the message passing capability of the pathway[2].Certainly,it is known that the sca?old will bind to a number of reagents which constitute successive steps in the pathway forming a complex in which the interacting reagents are in close proximity(see Figure1).

There have been some previous studies which looked at how the relative con-centrations of reagents,and the binding and release rates,a?ect the formation of protein complexes in general[3]and sca?old complexes in particular[4].At this early stage of our work we seek to recreate their results as much as anything, although the ultimate aim is to go beyond what has been done before.

Ferrell’s paper[2]proposes three implications of sca?old proteins:

1.too little sca?old leads to low signalling;

2.too much sca?old leads to low signalling;

2

Sca?old

000def

=(mapk

in

,k).Sca?old

100

+(mapkk

in

,k).Sca?old

010 +(mapkkk

in

,k).Sca?old

001

Sca?old

100def

=(mapk

out

,l).Sca?old

000

+(mapkk

in

,k).Sca?old

110 +(mapkkk

in

,k).Sca?old

101

Sca?old

010def

=(mapk

in

,k).Sca?old

110

+(mapkk

out

,l).Sca?old

000 +(mapkkk

in

,k).Sca?old

011

Sca?old

001def

=(mapk

in

,k).Sca?old

101

+(mapkk

in

,k).Sca?old

011 +(mapkkk

out

,l).Sca?old

000

Sca?old

110def

=(mapk

out

,l).Sca?old

010

+(mapkk

out

,l).Sca?old

100 +(mapkkk

in

,k).Sca?old

111

Sca?old

101def

=(mapk

out

,l).Sca?old

001

+(mapkk

in

,k).Sca?old

111 +(mapkkk

out

,l).Sca?old

100

Sca?old

011def

=(mapk

in

,k).Sca?old

111

+(mapkk

out

,l).Sca?old

001 +(mapkkk

out

,l).Sca?old

010

Sca?old

111def

=(mapk

out

,l).Sca?old

011

+(mapkk

out

,l).Sca?old

101 +(mapkkk

out

,l).Sca?old

110

MapK

free def

=(mapk

in

, ).MapK

bound

MapK

bound def

=(mapk

out

, ).MapK

free

MapKK

free def

=(mapkk

in

, ).MapKK

bound

MapKK

bound def

=(mapkk

out

, ).MapKK

free

MapKKK

free def

=(mapkkk

in

, ).MapKKK

bound

MapKKK

bound def

=(mapkkk

out

, ).MapKKK

free

Sca?old

000￡?

K

(MapK

free

MapKK

free

MapKKK

free

)

where K={mapk

in ,mapk

out

,mapkk

in

,mapkk

out

,mapkk k in,mapkkk

out

}

Figure2:Simple sca?old model to explore the dynamics of sca?olds and sub-strates

3.intermediate levels generate high signalling.

As a validation for our initial models we have addressed the question of how well we can recreate this behaviour?Moreover,we additionally consider the extent to which rates a?ect the outcomes.

3Simple model

Our initial model is shown in Figure2.In previous work[5]we have devel-oped PEPA models in two distinct style:reagent-centric and pathway-centric. In the reagent-centric style we focussed on the reagents in the pathway and assumed discrete levels of concentration.There is one set of component de?ni-tions for each reagent,each de?nition corresponding to one of the discrete levels of concentration.The component de?nition prescribes the actions(reactions) which can increase or decrease the concentration,therefore taking the reagent component to another state(level).In the extreme case this reduces to two

3

MapK

free def

=(mapk

in

,m1).MapK

bound

MapK

bound def

=(mapk

out

,m2).MapK

free

MapKK

free def

=(mapkk

in

,m3).MapKK

bound

MapKK

bound def

=(mapkk

out

,m4).MapKK

free

MapKKK

free def

=(mapkkk

in

,m5).MapKKK

bound

MapKKK

bound def

=(mapkkk

out

,m6).MapKKK

free

Figure3:Kinase components for Markovian model

parameter value

k1000

l1000

m1 1.0

m210.0

m3 1.0

m410.0

m5 1.0

m610.0

Table1:Parameter values for Markovian experiments

local states for the reagent corresponding to high and low concentration.In the pathway-centric style we focussed on subpathways,or cycles of behaviour within the model.The distinct components of the model correspond to those reagents which have initial concentrations,and the derivatives of the components are the di?erent states(e.g.phosphorylations,complex formations etc.)that the reagents may?nd themselves in.

For this model where we are interested in the structural rather than the signalling/concentration aspects of the system the reagent-centric style does not seem appropriate.Instead we adopt something closer to the pathway-centric style in which we focus on the possible state that the various components of the system may?nd themselves.The slight complication of this is that the ODE semantics has generally been applied to the models developed in the reagent-centric style[6].

The model we consider has a three position sca?old and three reagents,which are kinases,which may bind to it.Throughout these experiments we assume that signalling proceeds whenever the three reagents are bound,either in the sca?old(the only possibility here)or in the cytosol.

3.1Markovian experiments

When conducting Markovian analysis the structure of the model was as above but the passive rates of the kinases(MapK,MapKK,MapKKK)were replaced as shown below in Figure3

Di?erent con?gurations of the model were considered.For example,when there are three instances of the sca?old and three instances of each kinase and the release rate is faster than the binding rate then the con?guration of the

4

model is:

(Sca?old

000 Sca?old

000

Sca?old

000

)￡?L

(MapK MapKK MapKKK MapK MapKK

MapKKK MapK MapKK MapKKK)

where L={mapk

in

,mapk

out

,mapkk

in

,mapkk

out

,mapkkk

in

,mapkkk

out

and the rates are as shown in the Table1.

For a model with unlimited amounts of kinase we modify the corresponding part of the model as follows:

MapK def=(mapk

in ,m1).MapK+(mapk

out

,m2).MapK

MapKK def=(mapkk

in ,m1).MapKK+(mapkk

out

,m2).MapKK

MapKKK def=(mapkkk

in ,m1).MapKKK+(mapkkk

out

,m2).MapKKK

and the model con?guration becomes:

(Sca?old

000 Sca?old

000

Sca?old

000

)￡?L(MapK MapKK MapKKK)

A full sca?old is one in which all its positions are occupied,i.e.Sca?old111. Assume that the instances of sca?old are numbered1...n where n is the num-ber of sca?old instances.Note,sca?old instances are not to be confused with positions within a given sca?old instance.In the experiments,n=3.For each experiment we return the following results.

?The ratio of sca?old to kinase,written sca?old:kinase.The ratios corre-pond to equal amounts,more sca?old than kinase,and less sca?old than kinase.

?The likelihood(expressed as probability),in the steady state solution, of being in a state with sca?olds1,2,3full,sca?olds1,2full,and only sca?old1full.Call these probabilities p123,p12and p1,respectively.

?The total throughput,indicating that when a sca?old is full,a signalling event can occur.The throughput is calculated by summing the throughput for each of the cases:all sca?old instances full,two sca?old instances full, and one sca?old instance full.To compute these from the probabilities p123,p12and p1,we appeal to symmetry.Let comb i be the number of combinations of i sca?olds full.For example,assuming three sca?old instances,then comb1=3,comb2=3,comb3=1.

The formula for total throughput,for n sca?old instances,is given by:

(Σn i=0(p1...n×comb i×i))λ

whereλis the rate of the signalling event.

When n=3,the throughput is de?ned by:

((p123×3)+(p12×3×2)+(p1×3×1))λ

5

3.2Experiment set1:binding rate==release rate Consider the case where the binding and release rates are equal.

sca?old:kinase p123p12p1throughput

3:3.0019.125.015.80λ

3:20.0410?4.24λ

3:unlimited.015.037.125.64λ

It is clear that greatest signalling(i.e.throughput)occurs when the amounts of sca?old and kinase are equal.Least signalling occurs when there is too little kinase,but even when the kinase is unlimited,the throughput is still lower than in the case where they are equal.

3.3Experiment set2:binding rate>release rate

Now consider the case where the binding rate is an order of magnitude higher that the release rate.

sca?old:kinase p123p12p1throughput

3:3.42.75.00755.78λ

3:20.22.021.39λ

3:unlimited.052.125.321.87λ

Again,we see the same behaviour.Not suprisingly,the e?ect of equal amounts of sca?old and kinase is more pronounced when the binding rate is an order of magnitude higher;note that the di?erence between the other two cases(too much,too little kinase)is not signi?cant.

So,we can conclude that we see the expected behaviour.But this is only a rough impression,since the rates can only be relative and do not precisely implement known kinetics.The PEPA notation has been useful for formulating the model and the workbench for initial exploration,we now turn our atten-tion to interpretation via the continuous state space semantics,allowing us to conduct experiments with larger populations of molecules.

3.4Dizzy experiments

In order to analyse the models with larger populations of molecules,we used the tool written by Stephen Gilmore to translate a PEPA model into the format required for input to the Dizzy tool.

This is relatively straightforward but we found that the resulting.dizzy ?le needed some manipulation after the translation in order to get the rates of the synchronisation actions correct.This was because at di?erent parts of the model we found that we wanted di?erent kinetics in order to capture the correct rate of interaction between components.The tool written by Stephen was intended for computer science rather than systems biology case studies,and consequently uses the min-kinetics by default.

For the binding of kinase to sca?old we found that we needed mass action kinetics and so the Dizzy?le was edited to re?ect this,inserting the product

6

200

400

600

800

1000

1000 2000 3000

4000 5000 6000 7000 8000 9000 10000

N u m b e r o f f u l l s c a f f o l d s

Number of scaffolds

The effects of varying the amount of scaffold protein available

full scaffolds

No.of sca?olds

No.of reagents

Binding and No.of full sca?olds

Release rate

501000 1.049.81001000 1.099.72001000 1.01993001000 1.029******** 1.0398******* 1.04976001000 1.0596******* 1.06938001000 1.07889001000 1.0875******** 1.0910******** 1.080412001000 1.068413001000 1.058614001000 1.050615001000 1.044216001000 1.038917001000 1.034618001000 1.030719001000 1.027********* 1.024********* 1.016030001000 1.011135001000 1.081.540001000 1.062.410000

1000 1.0

10.0

Table 2:The e?ects of varying the amount of sca?old protein available (k =l =1.0)

7

of the relevant concentrations,as the rate constant multiplier for such actions. However for the release of a kinase from the sca?old,the min-kinetics was correct,as although they are represented apparently as separate entities in the PEPA model,the?lled sca?old and the bound kinase are really a single entity.

A number of experiments were carried out on this simple model to study the e?ect of varying the relative concentrations of sca?old and kinases,and the ratio of the binding and release rates.The results are shown in Tables2and3 respectively.

In Table2we can see the e?ect outlined by Ferrell[2].As we increase the number of sca?old proteins,keeping the number of kinases the same,we see the number of?lled sca?olds increasing but then decreasing again.Recall that signalling is deemed to occur in?lled sca?olds so we would expect a similar pattern for the signalling in the cell.The peak is reached when the number of sca?olds and kinases is balanced.This e?ect is sometimes termed combinatorial inhibition[4].

In Table3we see the e?ect of changing the ratio between the binding rate and the release rate.We assume in the current experiments that all the kinases have the same binding rate and the same release rate.Here we see that as the ratio increases,so that binding is more likely than release,so the number of ?lled sca?olds increases.

4Enhanced model

In this section we consider a model which also captures the possibility of a complex forming in the cytosol.This free-?oating complex formation is now in competition with the formation of a complex on the sca?old.This model is shown in Figure4.The sca?old aspect of the model remains unchanged but the MAPK reagents now have additional activities,re?ecting their ability to bind to each other,as well as binding to the sca?old.

4.1Dizzy experiments

Again we carried out experiments to investigate the impact of changing the concentration of the sca?old,and the ratio of the binding rate to the release rate(not shown here).We also now considered the impact of changing the concentration of the kinases.

As can be seen in Table4when the concentration of sca?old is varied we again see the characteristic shape of combinatorial inhibition.Note that now the peak is somewhat lower than in the previous experiment.This is because some of the kinases are binding in cytosol and are not available to enter the sca?old.

Table5shows the results of varying the concentration of one of the kinases, whilst keeping the concentration of sca?old protein and other kinases constant at1000.As the concentration of MapKK increases,we see an increase in the number of?lled sca?olds,but then a decrease again.This can be explained as the ability to form a sca?old with a full complement of kinases clearly depends on the availability of the kinases.However,as the concentration of MapKK increases,the ability to form complexes in the cytosol also increases(note that MapKK plays the central role in such complexes).Thus the number of full

8

740

760 780 800 820 840 860 880 900 920 940 0 0.2 0.4 0.6 0.8

1 1.

2 1.4 1.6 1.8 2

N u m b e r o f f u l l s c a f f o l d s

Binding rate

The effects of varying the ratio of the binding rate to the release rate with constant concentration

full scaffolds

No.of sca?olds

No.of reagents

Binding rate

Release rate

No.of full sca?olds 100010000.10 1.0741100010000.20 1.0809100010000.30 1.0841100010000.40 1.0861100010000.50 1.0874100010000.60 1.0885100010000.70 1.0893100010000.80 1.0899100010000.90 1.090510001000 1.0 1.0910******** 1.10 1.0914******** 1.20 1.0917******** 1.30 1.092010001000 1.40 1.092310001000 1.50 1.092510001000 1.60 1.092810001000 1.70 1.0930******** 1.80 1.0932******** 1.90 1.09341000

1000

2.0

1.0935

Table 3:The e?ects of varying the ratio of the binding rate to the release rate with constant concentration levels [Sca?old]=1000

9

Sca?old

000def

=(mapk

in

,k).Sca?old

100

+(mapkk

in

,k).Sca?old

010 +(mapkkk

in

,k).Sca?old

001

Sca?old

100def

=(mapk

out

,l).Sca?old

000

+(mapkk

in

,k).Sca?old

110 +(mapkkk

in

,k).Sca?old

101

Sca?old

010def

=(mapk

in

,k).Sca?old

110

+(mapkk

out

,l).Sca?old

000 +(mapkkk

in

,k).Sca?old

011

Sca?old

001def

=(mapk

in

,k).Sca?old

101

+(mapkk

in

,k).Sca?old

011 +(mapkkk

out

,l).Sca?old

000

Sca?old

110def

=(mapk

out

,l).Sca?old

010

+(mapkk

out

,l).Sca?old

100 +(mapkkk

in

,k).Sca?old

111

Sca?old

101def

=(mapk

out

,l).Sca?old

001

+(mapkk

in

,k).Sca?old

111 +(mapkkk

out

,l).Sca?old

100

Sca?old

011def

=(mapk

in

,k).Sca?old

111

+(mapkk

out

,l).Sca?old

001 +(mapkkk

out

,l).Sca?old

010

Sca?old

111def

=(mapk

out

,l).Sca?old

011

+(mapkk

out

,l).Sca?old

101 +(mapkkk

out

,l).Sca?old

110

MapK

free def

=(mapk

in

, ).MapK

sbound +(mapk

bind

,k).MapK

bound

MapK

sbound def

=(mapk

out

, ).MapK

free

MapK

bound def

=(mapk

unbind

,l).MapK

free

MapKK

free def

=(mapkk

in

, ).MapKK

sbound +(mapk

bind

, ).MapKK

kbound +(mapkk

bind

,k).MapKK

bound

MapKK

kbound def

=(mapk

unbind

, ).MapKK

free

MapKK

bound def

=(mapkk

unbind

,l).MapKK

free

MapKK

sbound def

=(mapkk

out

, ).MapKK

free

MapKKK

free def

=(mapkkk

in

, ).MapKKK

bound +(mapkk

bind

, ).MapKKK

kbound

MapKKK

bound def

=(mapkkk

out

, ).MapKKK

free

MapKKK

kbound def

=(mapkk

unbind

, ).MapKKK

free

Sca?old

000￡?

L

(MapK

free

￡?

M

MapKK

free

￡?

M

MapKKK

free

)

where L={mapk

in ,mapk

out

,mapkk

in

,mapkk

out

,mapkkk

in

,mapkkk

out

}

and M={mapk

bind ,mapk

unbind

}

Figure4:More complex sca?old model to explore the dynamics of sca?olds and substrates including the binding between free-?oating MapK,MapKK and MapKKK elements

10

100 200 300 400 500 600 700 800 0 1000 2000 3000

4000 5000 6000 7000 8000 9000 10000

N u m b e r o f f u l l s c a f f o l d s

Number of scaffolds

The effects of varying the amount of scaffold protein available

full scaffolds

No.of sca?olds

No.of reagents

Binding and No.of full sca?olds

Release rate

501000 1.025.51001000 1.052.32001000 1.01103001000 1.01754001000 1.024******** 1.03286001000 1.0417******* 1.0516******* 1.06159001000 1.069410001000 1.072511001000 1.0701******** 1.063813001000 1.0564******** 1.049515001000 1.0435******** 1.038517001000 1.034218001000 1.030619001000 1.027********* 1.024********* 1.015930001000 1.011135001000 1.081.540001000 1.062.410000

1000 1.0

10.0

Table 4:The e?ects of varying the amount of sca?old protein available (k =l =1.0,r =s =1.0)

11

100 200 300 400 500 600 700 800 0 500

1000 1500 2000 2500

N u m b e r o f f u l l s c a f f o l d s

Number of reagent MAPKK

The effects of varying the concentration of MAPKK with constant scaffold concentration levels

full scaffolds

No.of sca?olds,No.of reagent Binding and No.of full sca?olds

MAPK and MAPKKK

MAPKK Release rate

1000100 1.087.71000200 1.01741000300 1.025******** 1.03421000500 1.04221000600 1.04991000700 1.0571******* 1.06351000900 1.068910001000 1.072510001100 1.0737******** 1.0722******** 1.068510001400 1.063810001500 1.058710001600 1.0539******** 1.049310001800 1.0451******** 1.0412******** 1.0378******** 1.034610002200 1.0318******** 1.029********* 1.026910002500 1.0

248

Table 5:The e?ects of varying the concentration of MAPKK with constant sca?old concentration levels [Sca?old]=1000

12

No gnuplots available yet.....

No.of sca?olds,binding release binding release No.of full and kinases rate k(S)rate l(S)rate r(C)rate s(C)sca?olds 1000 1.0 1.0 1.0 1.0725

1000 2.0 1.0 1.0 1.0814

1000 3.0 1.0 1.0 1.0853

1000 4.0 1.0 1.0 1.0877

1000 5.0 1.0 1.0 1.0892

1000 2.0 2.0 1.0 1.0725

1000 3.0 3.0 1.0 1.0725

1000 4.0 4.0 1.0 1.0725

1000 5.0 5.0 1.0 1.0725

1000 1.0 1.0 2.0 1.0671

1000 1.0 1.0 3.0 1.0636

1000 1.0 1.0 4.0 1.0610

1000 1.0 1.0 5.0 1.0588

1000 1.0 1.0 2.0 2.0725

1000 1.0 1.0 3.0 3.0725

1000 1.0 1.0 4.0 4.0725

1000 1.0 1.0 5.0 5.0725 Table6:The e?ects of varying the binding/release rates with constant sca?old and kinase concentration levels[Sca?old]=1000

sca?olds declines as the amount of MapKK continues to increase.Interestingly the peak value for full sca?olds occurs when there is slightly more of MapKK than the other reagents(1200).

In a further set of experiments we investigate the impact of varying the rates of binding in the sca?old with respect to the rate of binding in the cy-tosol,and also the rate of both forms of binding with respect to the rate of the corresponding release.These results are shown in Table6.

The signi?cant factor seems to be the ratio of the ratios between the binding and release rates in the sca?old(denoted(S))and the cytosol(denoted(C)) respectively.

5Conclusions and further work

So far our experiments have only been preliminary and to some extent they replicate those already reported in the literature[3,4].We have focussed on the formation of the sca?old complex.In future work we plan to look beyond the sca?old complex to its role within the signalling pathway,and investigate the implications of the ability of the sca?old to increase the throughput of the pathway.

13

References

[1]D.Morrison and R.Davis,“Regulation of MAP kinase signalling modules by

sca?old proteins in mammals,”Annual Review of Cell Development Biology, vol.19,pp.91–118,2003.

[2]J.Ferrell,“What do sca?old proteins really do?,”Science’s STKE,pp.1–3,

2000.

[3]D.Bray and https://www.wendangku.net/doc/234703985.html,y,“Computer-based analysis of the binding steps in pro-

tein complex formation,”Proceedings of the National Academy of Science, U.S.A.,vol.94,pp.13493–13498,1997.

[4]A.Levchenko,J.Bruck,and P.Sternberg,“Sca?old proteins may bipha-

sically a?ect the levels of mitogen-activated protein kinase signalling and reduce its threshold properties,”Proceedings of the National Academy of Science,U.S.A.,vol.97,pp.5818–5823,May2000.

[5]M.Calder,S.Gilmore,and J.Hillston,“Modelling the in?uence of RKIP

on the ERK signalling pathway using the stochastic process algebra PEPA,”

Transactions on Computational Systems Biology,2006.to appear.

[6]M.Calder,S.Gilmore,and J.Hillston,“Automatically deriving ODEs from

process algebra models of signalling pathways,”in Proceedings of Computa-tional Methods in Systems Biology2005,pp.204–215,2005.

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初中英语语法知识—动词的分类汇编

一、选择题 1．Karen and Helen _______ my brother's friends. I know _______. A．is; her B．are; them C．are; her D．is; them 2．Kay looked _________ at the guests who said that the food she cooked tasted _________. A．happily; wonderfully B．happily; wonderful C．happy; wonderful D．happy; wonderfully 3．I don’t have a baseball, but Alan A．do B．does C．have D．has 4．—Listen! I can hear someone __________ for help. —Is there __________? A．calling; anything wrong B．call; anything wrong C．calling; wrong anything D．call; wrong anything 5．Our school basketball team ________ in the final of the basketball competition. Another team from No.5 Middle School ________ doing very well too. A．is; isn’t B．are; aren’t C．is; are D．are; is 6．—Why ______ you so busy these days? —Because they arrived ______ London ______ the morning of July1. A．are; in , in B．are; in , on C．do; on , in D．do; at , on 7．A number of visitors ________visiting our school. The number of them________ about 180. A．is;are B．are; is C．is; is D．are; are 8．—William, your hat _______ nice. —Thanks. A．buys B．looks C．finds 9．This is a photo of my grandpa. He young A．looks B．feels C．sounds D．hears 10．Lily and Lucy _______ their mother. They have big eyes and yellow hair. A．like both B．both are like C．both like D．are both like 11．—He’s never been late for school. —________________. A．So have I B．So am I C．Neither have I D．Nor am I 12．In the past he often made his sister____, but now he is often made ___by his sister. A．to cry; to cry B．cry; cry C．to cry; cry D．cry; to cry 13．This my sister and those my brothers. A．is, is B．are, are C．is, are 14．— Tom in the library? —Yes,and his friends Eric and Dale in the library,too. A．Is;are B．Is;is C．Are;is

初中英语语法知识—动词时态的分类汇编及解析

一、选择题 1．—Remember the first time we met, Jim? —Of course I do. You ________ in the library. A．were reading B．have read C．will read D．read 2．— How about going for a drive, Mike? — One moment, please! I __________ cleaning our room soon. A．will finish B．have finished C．finish D．finishes 3．I________ cleaning my room.It’s clean now. A．have finished B．finished C．finish D．will finish 4．It ________ heavily when I left the movie theater. A．rains B．was raining C．is raining D．will rain 5．Dick __________ in America, but he has been ___________ Chinese food since he moved to China. A．used to live; used to eating B．is used to live; used to eat C．is used to live; used to eating D．used to living; used to eat 6．—I don’t understand why you didn’t go to the lecture yesterda y afternoon. —I’m so sorry. But I my homework. A．had done B．was doing C．would do D．am doing 7．Sorry, Jenny isn’t here. She________ up some lights in the garden. A．put B．puts C．is putting D．has put 8．—I'm sorry. There was too much traffic on the road. —Never mind. The meeting________for only 5 minutes. A．has begun B．has stopped C．has been on 9．The Great Wall (长城) ________ long and it ________ a long history (历史). A．has; is B．is; is C．is; has D．have; has 10．They the English role play for the show last night. A．will practice B．practiced C．is practicing D．have practiced 11．— Look after yourself on your way to Gungzhou,dear. — OK, mom.I will call you________get there. A．as soon as B．so that C．because 12．—Surprise! Cindy is singing in the concert. —But she ______. A．will refuse B．refused C．refuses D．has refuse 13．It’s _______today．I think it’s going to________． A．cloud，rain

新初中英语语法知识—动词的分类汇编附解析

一、选择题 1．Tom and Jim __________ in Class 3. A．is B．be C．am D．are 2．— ________ your cousin and her friends like history? —Yes, they ________ it’s very interesting. A．Does; think B．Do ; think C．Do; thinks 3．My mother with me ________ at my grandma’s home ________that snowy night. A．were, at B．was, at C．was, on 4．My parents don’t_______me eat_______junk food. A．like; too many B．want; too much C．ask; too many D．let; too much 5．I tried to make the baby ________by singing, and that worked well at last. A．stop crying B．to stop crying C．stop to cry D．to stop to cry 6．—How much ________ this pair of shoes? —Five dollars. A．am B．is C．are D．be 7．—William, your hat _______ nice. —Thanks. A．buys B．looks C．finds 8．Which of the following sentences is right? A．My books on the desk B．Nice meet you, too C．These are my dictionaries D．Thanks you for your help. 9．—Hey, Jack. Do you want to go to the library? —That ________ great. I love reading. A．hears B．looks C．sounds D．listens 10．—The books on the table_________ Mike’s, r ight? —Yes, they are. A．am B．is C．are 11．This my sister and those my brothers. A．is, is B．are, are C．is, are 12．—________ Mary________ fruit? —Of course. A．Is; like B．Does; likes C．Does; like D．Do; likes 13．Karen and Helen _______ my brother's friends. I know _______. A．is; her B．are; them C．are; her D．is; them 14．— Tom in the library? —Yes,and his friends Eric and Dale in the library,too. A．Is;are B．Is;is C．Are;is 15．— When and where shall we meet? — Let's ________it half past nine.

初中英语语法复习：动词的分类

2010年中考英语汇总--动词的分类概念：表示动作中状态的词叫做动词。 动词的分类 1.按照在句子中的功能，动词可分为四类：实义动词、系动词、助动词、情态动词。 说明：某些情况下，有些动词既可做实义动词，又可做助动词，例如： We are having a meeting.我们正在开会。（having是实义动词。） He has gone to New York.他已去纽约。（has是助动词。） 2. 按照后面是否有宾语，分为两类：及物动词（vt）、不及物动词（vi）。 说明：同一动词有时可用作及物动词，有时可用作不及物动词。例如： She can dance and sing.她能唱歌又能跳舞。（sing在此用作不及物动词。） She can sing many English songs. 她能唱好多首英文歌曲。（sing用作及物动词。） 3. 根据是否受主语的限制，分两类：限定动词、非限定动词。例如： She sings very well. 她唱得很好。（sing受主语she的限制，故用第三人称单数形式sings.） She wants to learn English well. 她想学好英语。（to learn不受主语she的限制，没有词形变化，是非限定动词。） 说明：英语中共有三种非限定动词，分别是：动词不定式、动名词、分词。 在这里我们多说一下to+动词原型，和ing形式。英语不同于我们的汉语，不可以两个动词联用。如，我们汉语说，我喜欢吃苹果。其中“喜欢”和“吃”是两个动词，汉语这样说没有问题，如果翻译成英语，就变成了“I like eat apple.”这种说法按照英语的规定是不对的。那怎么办呢，于是就在两个动词之间多了一个“to”，或者把第二个词加上ing.于是就变成了“I like to eat apple.或者I like eatting apple”这样说来，动词是不是很简单啦！ 4.根据动词的组成形式，可分为三类：单字词、短语动词、动词短语。例如： The English language contains many phrasal verbs and verbal phrases. 英语里有许多短语动词和动词短语。（contains是单字动词。） Students should learn to look up new words in dictionaries. 学生们学会查字典。（look up是短语动词。） The young ought to take care of the old. 年轻人应照料老人。（take care of是动词短语。） 5.动词有五种形态：原形、第三人称单数形式、过去式、过去分词、现在分词。

初中英语语法知识—动词时态的分类汇编(1)

一、选择题 1．Look! Dave __________ on the phone. A．talks B．are talking C．talk D．is talking 2．—Surprise! Cindy is singing in the concert. —But she ______. A．will refuse B．refused C．refuses D．has refuse 3．—Rose, can you give me a hand? —Just a minute. I ______ the followers. A．am watering B．have watered C．watered D．water 4．— How about going for a drive, Mike? — One moment, please! I __________ cleaning our room soon. A．will finish B．have finished C．finish D．finishes 5．Come on, John! You are too slow! Look, the parade _________ for fifteen minutes! A．started B．has started C．has been on D．has been open 6．No noise, please. Your brother ________ his homework in the next room. A．does B．is doing C．did D．has done 7．It ________ heavily when I left the movie theater. A．rains B．was raining C．is raining D．will rain 8．Dick __________ in America, but he has been ___________ Chinese food since he moved to China. A．used to live; used to eating B．is used to live; used to eat C．is used to live; used to eating D．used to living; used to eat 9．Monica, you ______ the exam! Congratulation! A．pass B．have passed C．will pass D．are passing 10．Sorry, Jenny isn’t here. She________ up some lights in the garden. A．put B．puts C．is putting D．has put 11．—How much is the ticket (票) to Central Park? —One ticket $40, and you can $80 for two persons. A．costs; pay B．cost; spend C．pay; spend D．spends; pay 12．—Look, Tom's parents look so sad. —Maybe they what's happened. A．knew B．have known C．has known D．will know 13．Nick's brother________the navy in 2011and ________the navy since then.

初中英语语法知识—动词的分类汇编及答案(4)

一、选择题 1．—Do you know whom they will have ________the parts of car together?—Sorry,I have no idea. A．fix B．fixed C．to fix D．fixing 2．Coffee is ready. How nice it ______! Would you like some? A．looks B．smells C．sounds D．feels 3．I don’t have a baseball, but Alan A．do B．does C．have D．has 4．—Listen! I can hear someone __________ for help. —Is there __________? A．calling; anything wrong B．call; anything wrong C．calling; wrong anything D．call; wrong anything 5．— ________ your cousin and her friends like history? —Yes, they ________ it’s very interesting. A．Does; think B．Do ; think C．Do; thinks 6．—Why ______ you so busy these days? —Because they arrived ______ London ______ the morning of July1. A．are; in , in B．are; in , on C．do; on , in D．do; at , on 7．A number of visitors ________visiting our school. The number of them________ about 180. A．is;are B．are; is C．is; is D．are; are 8．This is a photo of my grandpa. He young A．looks B．feels C．sounds D．hears 9．—When you free last week? — Well, I have a free day because there was too much work. A．did; didn’t B．were; didn’t C．were; wasn’t D．did; wasn’t 10．—Where_______you come from? —I______from Japan A．are; am B．are; come C．do; come 11．________he_______big________? A．Does; has; foot B．Does; have; feet C．Do; have; foots 12．In the past he often made his sister____, but now he is often made ___by his sister. A．to cry; to cry B．cry; cry C．to cry; cry D．cry; to cry 13．—________ you Mary? —Yes, I ________. A．Are; is B．Is; am C．Are; am D．Am; is 14．—Do you have an art festival at school?

中考英语语法复习：动词的分类

中考英语语法复习：动词的分类 动词的分类 一、知识要点 1.及物动词和不及物动词 根据动词在句子中是否可以接宾语，可以把动词分为及物动词和不及物动词两种类型： A.能接宾语的动词称为及物动词，可分为跟单宾语的及物动词、跟双宾语的及物动词、跟复合宾语(即宾语+宾语补足语)的及物动词等。 My brother is flying the kite on the playground. 我的弟弟正在操场上放风筝。(fly 后跟单宾语kite) My mother gives me a new bike as the birthday present. 我妈妈给我一辆新自行车作为生日礼物。(give后跟双宾语me和a new bike) The teacher made his students happy by doing some games. 老师通过做游戏使得学生高兴。(made后跟宾语his students和宾语补足语happy) 注意1：有些双宾语动词间接宾语(人)放在直接宾语(事物)后时，间接宾语前要加to。常用的此类词有bring、give、hand、pass、pay、post、return、sell、show、teach、tell、throw、lend等。 Hand me that book, please. = Hand that book to me, please. 请递给我那本书。 有些双宾语动词间接宾语(人)放在直接宾语(事物)后时，间接宾语前要加for。常用的此类词有buy、choose、cook、draw、book、find、get、make、order等。 My mom bought me a nice backpack. = My mom bought a nice backpack for me. 我妈妈为我买了一个好看的书包。

初中英语语法知识—动词的分类汇编(5)

一、选择题 1．________he_______big________? A．Does; has; foot B．Does; have; feet C．Do; have; foots 2．Kay looked _________ at the guests who said that the food she cooked tasted _________. A．happily; wonderfully B．happily; wonderful C．happy; wonderful D．happy; wonderfully 3．Parents always hope their children a happy and healthy life. A．to live B．can live C．living D．should live 4．The woman made her husband_______ outside the gate yesterday afternoon. A．wait B．waiting C．to wait D．waited 5．I don’t have a bas eball, but Alan A．do B．does C．have D．has 6．My parents don’t_______me eat_______junk food. A．like; too many B．want; too much C．ask; too many D．let; too much 7．The library ______ from 8 a.m. to 3 p.m. on weekdays. But it______ at 5 p.m. on Saturdays and Sundays. A．is open; closes B．opens; closed C．is opened; closes D．is opening; is closed 8．This is a photo of my grandpa. He young A．looks B．feels C．sounds D．hears 9．Lily and Lucy _______ their mother. They have big eyes and yellow hair. A．like both B．both are like C．both like D．are both like 10．—When you free last week? — Well, I have a free day because there was too much work. A．did; didn’t B．were; didn’t C．were; wasn’t D．did; wasn’t 11．—He’s never been late for school. —________________. A．So have I B．So am I C．Neither have I D．Nor am I 12．—Do you like watching cooking programs on TV? —No, I don’t , but my twin brother . He's very fond of cooking. A．does B．do C．is D．are 13．—Where_______you come from? —I______from Japan A．are; am B．are; come C．do; come 14．— Tom in the library? —Yes,and his friends Eric and Dale in the library,too. A．Is;are B．Is;is C．Are;is 15．— What kind of home do you ? — A flat three bedrooms.

初中英语语法知识—动词时态的分类汇编(2)

一、选择题 1．Don’t turn on the TV. Grandma ________. A．sleeps B．is sleeping C．sleep D．are sleeping 2．—Listen！Who in the music room? —It must be Sally. She there every day. A．sings, sings B．is singing, sings C．sings, is singing 3．—Surprise! Cindy is singing in the concert. —But she ______. A．will refuse B．refused C．refuses D．has refuse 4．—Rose, can you give me a hand? —Just a minute. I ______ the followers. A．am watering B．have watered C．watered D．water 5．—Mom, can you teach me how to pronounce the words? —Wait a minute. I ________. A．am cooking B．cook C．cooked D．will cook 6．No noise, please. Your brother ________ his homework in the next room. A．does B．is doing C．did D．has done 7．It ________ heavily when I left the movie theater. A．rains B．was raining C．is raining D．will rain 8．It’s 4:00 p.m. The students _____ in the pool. A．swim B．swam C．swims D．are swimming 9．—________ a remake (翻拍) of the famous Disney movie Lion King in July, 2019.— Really? I can't wait to see it. A．There will have B．There is going to have C．There will be 10．—I don’t understand why you didn’t go to the lecture yesterday afternoon. —I’m so sorry. But I my homework. A．had done B．was doing C．would do D．am doing 11．—How much is the ticket (票) to Central Park? —One ticket $40, and you can $80 for two persons. A．costs; pay B．cost; spend C．pay; spend D．spends; pay 12．John and Mary ________ good eating habits but Eric ________. A．have; don’t B．has; doesn’t C．have; doesn’t D．has; don’t 13．They the English role play for the show last night. A．will practice B．practiced C．is practicing D．have practiced 14．When I was young, my mother ___________ by my side all the time.

初中英语语法知识—动词的分类汇编及答案(1)

一、选择题 1．—He’s never been late for school. —________________. A．So have I B．So am I C．Neither have I D．Nor am I 2．Colours can change our moods and make us happy or sad . A．feel B．to feel C．felt D．feeling 3．Kay looked _________ at the guests who said that the food she cooked tasted _________. A．happily; wonderfully B．happily; wonderful C．happy; wonderful D．happy; wonderfully 4．The woman made her husband_______ outside the gate yesterday afternoon. A．wait B．waiting C．to wait D．waited 5．Jim a basketball? A．Does; have B．Does; has C．Is; have 6．I don’t have a baseball, but Alan A．do B．does C．have D．has 7．What kind of music ________ he ________? A．does; listen B．does; listen to C．is; listen D．is; listen to 8．My parents don’t_______me eat_______junk food. A．like; too many B．want; too much C．ask; too many D．let; too much 9．—_______everyone get tired today? —Yes, we_______very tired the whole afternoon. A．Does; are all B．Do; are all C．Does; all are D．Is; all are 10．—Let’s _________ the library thi s afternoon. —Sorry, I want _________ my grandfather. A．go to;to meet B．go to;meeting C．to go to;to meet 11．The library ______ from 8 a.m. to 3 p.m. on weekdays. But it______ at 5 p.m. on Saturdays and Sundays. A．is open; closes B．opens; closed C．is opened; closes D．is opening; is closed 12．Many students are so crazy about mobile games that they lose interest in their schoolwork, which _________ their parents and teachers a lot. A．worries about B．is worried about C．worries D．is worried 13．Which of the following sentences is right? A．My books on the desk B．Nice meet you, too C．These are my dictionaries D．Thanks you for your help. 14．Mike and Jeff________ TV on weekdays.

新初中英语语法知识—动词的分类汇编(1)

一、选择题 1．This is a photo of my grandpa. He young A．looks B．feels C．sounds D．hears 2．In the past he often made his sister____, but now he is often made ___by his sister. A．to cry; to cry B．cry; cry C．to cry; cry D．cry; to cry 3．—Do you like watching cooking programs on TV? —No, I don’t , but my twin brother . He's very fond of cooking. A．does B．do C．is D．are 4．Colours can change our moods and make us happy or sad . A．feel B．to feel C．felt D．feeling 5．—________ Mary________ fruit? —Of course. A．Is; like B．Does; likes C．Does; like D．Do; likes 6．Here _______a nice photo of my family. A．am B．be C．is D．are 7．Parents always hope their children a happy and healthy life. A．to live B．can live C．living D．should live 8．Our school basketball team ________ in the final of the basketball competition. Another team from No.5 Middle School ________ doing very well too. A．is; isn’t B．are; aren’t C．is; are D．are; is 9．My mother with me ________ at my grandma’s home ________that snowy night. A．were, at B．was, at C．was, on 10．—_______everyone get tired today? —Yes, we_______very tired the whole afternoon. A．Does; are all B．Do; are all C．Does; all are D．Is; all are 11．The library ______ from 8 a.m. to 3 p.m. on weekdays. But it______ at 5 p.m. on Saturdays and Sundays. A．is open; closes B．opens; closed C．is opened; closes D．is opening; is closed 12．Hard work can bring you , but if you are lazy, you won’t become . A．success; success B．successful; success C．success; successful D．successful; succeed 13．—William, your hat _______ nice. —Thanks. A．buys B．looks C．finds 14．—How much ________ this pair of socks? —Two dollars for one pair.

初中英语语法知识—动词的分类汇编含答案(3)

一、选择题 1．What a beautiful song! It ___________so sweet. A．feels B．sounds C．tastes D．smells 2．Coffee is ready. How nice it ______! Would you like some? A．looks B．smells C．sounds D．feels 3．I don’t have a baseball, but Alan A．do B．does C．have D．has 4．What kind of music ________ he ________? A．does; listen B．does; listen to C．is; listen D．is; listen to 5．Lucy and I ________ good friends. A．am B．is C．are 6．A number of visitors ________visiting our school. The number of them________ about 180. A．is;are B．are; is C．is; is D．are; are 7．—What will the weather _______tomorrow? — It is going to_______ . A．be like; rainy B．be like; rain C．like; rain D．like; rainy 8．—How much ________ this pair of shoes? —Five dollars. A．am B．is C．are D．be 9．Which of the following sentences is right? A．My books on the desk B．Nice meet you, too C．These are my dictionaries D．Thanks you for your help. 10．—When you free last week? — Well, I have a free day because there was too much work. A．did; didn’t B．were; didn’t C．wer e; wasn’t D．did; wasn’t 11．The shopping mall near our school _________ for 12 hours a day. It _________ at 9:00 p.m.. A．opens; closes B．opens; is closed C．is open; is closed D．is open; closes 12．The woman made her husband_______ outside the gate yesterday afternoon. A．wait B．waiting C．to wait D．waited 13．—Hey, Jack. Do you want to go to the library? —That ________ great. I love reading. A．hears B．looks C．sounds D．listens 14．Mike and Jeff________ TV on weekdays. A．doesn't watch B．don't watch C．watches D．not watch 15．This pair of pants______gray. What color________your pants? A．are; are B．is;is C．is;are 16．________he_______big________? A．Does; has; foot B．Does; have; feet C．Do; have; foots 17．—Would you like to try some pizza?