From EST analysis to potential silk gene discovery 1

76 Table 4.6 Most abundant cDNA clones 78 Table 5.1 Amino acid composition of tubuliform silk and the silk genes identified from the tubuliform gland 148... Unlike other silk proteins, t

From EST analysis to potential silk gene discovery

From EST analysis to potential silk gene discovery

HUANG WEIDONG (B.Sc., M.Sc.)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE

2006

Acknowledgements

I would like to express my appreciation to my supervisor, Associate Professor Yang Daiwen and Professor Lam Toong Jin, for their invaluable guidance, patience, and trust throughout the project

Special thanks to my former supervisor, Associate Professor Sin Yoke Min Without his encouragement it will not have been possible for me to complete this project I would also like to express my sincere appreciation to Associate Professor Gong Zhiyuan, for his helpful advice and critical suggestions

I would also like to thank all my colleagues, including some former lab members, for their friendship, help and company Special thanks to Ashok Hegde, Seng Eng Kuan, Lin Zhi, Zhang Wensheng and Wu Qiang

The financial assistance in the form of a research scholarship provided by NUS is gratefully acknowledged

I would also like to thank my parents for their sustaining love Although words are not even enough to express my gratitude, I would still like to thank my husband Jiayi, without his constant love and support, I would not have been able to accomplish or even start this thesis

Table of Contents

Reference

Appendix

Chapter 1 General introduction 3

2.2.3 Mechanical properties of spider silks 15

2.3.1 Identification of spider silk genes 16

2.3.2 Structure and organization of spider silk genes 23

2.3.2.1 Spider silk genes are large transcripts 23

2.3.2.2 Spider silk genes contain internal repetitive sequences 24 2.3.2.3 Spider silk genes have a C-terminal conservative region 26 2.3.2.4 Codon usage analysis and bias of spider silk genes 27

2.4 Expression and structural analysis of spider silk proteins 29 2.4.1 Expression of spider silk proteins in vitro 29 2.4.2 Structural analysis of spider silk proteins 32

2.5 EST strategy: history, applications and further trend 37

2.5.2 Characteristics and analysis of EST data 38

2.5.3.4 Gene annotation from genomic sequences 43

3.2.2 Preparation of plasmid DNA from E coli 47

3.2.3 DNA digestion, DNA fragment purification and ligation 48

3.4.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 52

3.4.4 Desalting and buffer exchange of proteins 53

3.5.1 Preparation of E coli competent cells used

for heat-shock transformation 54

3.5.2 Transformation of E.coli competent cells 55

Chapter 4 cDNA library construction, generation and analysis of EST clones

4.3.1.1 Zap- cDNA synthesis 59

4.3.1.5 Preservation of Spider Silk cDNA library 64 4.3.2 Generation and analysis of cDNA clone 64

4.4.1 Construction and characterization of spider silk glands

4.4.5 Novel cDNA clones containing repetitive sequence 84

5.3.2.4 Hybridization and immunological detection 106

5.3.2.6 Probe removal from Northern blots 107 5.3.3 Expression specificity analysis (mRNA level) of TuSp1 107

5.3.3.2.4.1 RNA probe synthesis 109

5.3.3.2.5.1 Deparaffination and rehydration 111 5.3.3.2.5.2 Proteinase K treatment and post fixation 112

5.3.4 Expression specificity analysis (protein level) of TuSp1 114

5.3.4.1 Recombinant protein expression of TuSp1 114

5.3.4.2 Generation of antiserum against TuSp1 115

5.3.4.4 Immunofluorescence staining analysis 117 5.3.5 Structural analysis of recombinant protein Tu-81 118

5.4.3 Expression specificity of TuSp1 (mRNA level) 126

5.4.4 Expression specificity of TuSp1 (protein level) 137 5.4.4.1 Expression of TuSp1 recombinant proteins 137

5.4.5 TuSp1 is the major component in tubuliform gland and

it encodes a novel fibroin gene from Nephila antipodiana 147

Chapter 6 General discussion and conclusion 160

Reference 169

List of Figures

Figure 2.1 The seven specialized glands and their different

amino acid compositions of a typical Araneid orb weaver 9 Figure 2.2 Spider spinneret (silk secreted from piriform gland spigot,

Spiny Back Spider, Castercantha sp.) 12 Figure 2.3A Diagrammatic representation of spider spinneret 13 Figure 2.3B Single spider spinneret showing the internal anatomy

Figure 2.5 Predicted amino acid sequence for the Spidroin 2 protein,

rearranged to show repetitive elements 24 Figure 2.6 The silk proteins from N clavipes are depicted as generalized,

or consensus, amino acid repeats Subscripts indicate the number of times a sequence is tandemly repeated 25 Figure 4.1 Nucleotide and deduced amino acid sequences of EST clone

Figure 4.2 Nucleotide and deduced amino acid sequences of the EST clone

B6 87 Figure 4.3 Nucleotide and deduced amino acid sequences of EST clone

C622 88 Figure 4.4 Nucleotide and deduced amino acid sequences of EST clone

C675 89

Figure 4.5 Nucleotide and deduced amino acid sequences of EST clone

D106 90 Figure 4.6 Nucleotide and deduced amino acid sequences of EST clone

Figure 4-9 RT-PCR to analyze the mRNA levels of EST clone B6 96

Figure 4-10 SDS-PAGE to analyze the recombinant protein of A105, B386

Figure 5.1 Nucleotide and deduced amino acid sequences of the cDNA of

Figure 5.2 Schematic representation of the domain structure and internal

repetitive sequence of TuSp1 123 Figure 5.3 Northern blot analysis to show the expression of TuSp1

in Nephila antipodiana 125

Figure 5.4 RT-PCR to analyze the expression of TuSp1 (clone 81) 128

Figure S1 Probe—against MiSp1 from young female spiders 133 Figure S2 Probe—against TuSp1 from young female spiders 134 Figure S3 Probe—against MiSp1 from mature female spiders 135 Figure S4 Probe—against TuSp1 from mature female spiders 136

Figure 5.6 SDS-PAGE to analyze the recombinant protein of 81-1a

Figure 5.11 Alignment of spider silk fibroin C-terminal amino acid sequences 150

Figure 5.13 The CD and 1D NMR spectrum of TuSp1-1a 154

List of Tables

Table 2.1 Mechanical properties of different materials 15 Table 2.2 Summary the information of the identified silk genes 22 Table 2.3 Summary of the size of the typical silk fibroin genes 24 Table 2.4 Summary of the codon usage data of the most abundant

amino acids from the spider fibroin genes 27

Table 4.2 Primer pairs used for recombinant protein synthesis 69

Table 4.4 Summary of spider silk glands EST clones 74 Table 4.5 Distribution of identified EST clones in the cDNA library 76 Table 4.6 Most abundant cDNA clones 78 Table 5.1 Amino acid composition of tubuliform silk and the silk genes

identified from the tubuliform gland 148

Carboxy-terminal Dalton (kilodalton) Deoxyribonucleic acid Digoxigenin

Dithiothreitol Ethylenediaminetetraacetic acid Gram

Isopropyl-β-D-1-thiogalactopyranoside Kilobase pairs

Liter Luria Bertani Messager RNA open reading frame Microgram

Microliter Micromolar Milligram Milliter Minute Molar Nanogram Polyacrylamide gel electrophoresis Polymerase chain reaction

Ribonucleic acid Seconds

Sodium dodecyl sulphate Standard saline citrate Tris-acetate EDTA Tris-borate EDTA Tris-EDTA Weight per volume Ultraviolet

Voltes Volume per volume

Summary

Spider silk is renowned for its excellent mechanical properties: a balanced combination of high tensile strength and elasticity makes it one of the toughest materials of its kind In our present study, a cDNA library from a pool of all seven silk glands from a

local tropical species, Nephila antipodiana, was constructed and an expressed sequence tag

(EST) approach was used to isolate genes involved in spider silk production

In summary, >3000 random clones were examined for insert size by PCR and approximately 1000 clones with inserts of 1 kb were selected for sequencing Using

Northern blot analysis, RT-PCR reactions, in situ hybridization and Western blot analysis,

a novel silk cDNA clone from the golden web spider Nephila antipodiana was analyzed It

is serine rich and the partial cDNA fragment contains two almost identical fragments with one conserved spider fibroin-like C-terminal domain We demonstrate experimentally that this molecule is specifically expressed in the tubuliform gland and likely encodes one fibroin protein from the tubuliform gland, which supplies the main component of the inner egg case

Unlike other silk proteins, the protein encoded by the novel cDNA in water solution exhibits the characteristic of an α-helical protein, though the solid fiber of spider cocoon silk (produced in tubuliform gland) is mainly constituent of β sheet structure The unique structure implies the distinct property of the egg case silk Its sequence information facilitates elucidation of the evolutionary history of the araneoid fibroin genes

Thus we have obtained a novel partial cDNA of the spider fibroin gene family In addition, some other clones with novel repetitive amino acid sequences are described They may provide to be targets for further protein sequence, structure and functional studies

Chapter 1 General introduction

The golden-web weaver, Nephila spp., is known to produce up to seven different

silks from different types of abdominal glands of the spider (Foelix, 1996) Because of the excellent mechanical properties of the spider silks, they have become the subject for intensive studies in the last couple of years

A number of spider fibroin cDNA\genes have been identified, including the major ampullate fibroin MaSp1 and MaSp2 (Xu and Leiws, 1990; Hinman and Lewis, 1992;

Beckwitt and Arcdiacono, 1994; Guerette et al., 1996; Beckwitt et al., 1998; Gatesy et al., 2001); the minor ampullate silk gland fibroin MiSp1 and MiSp2 (Guerette et al., 1996;

Colgin and Lewis, 1998); the flagelliform silk gland fibroin Flag (Hayashi and Lewis, 1998;

Gatesy et al., 2001), the tubliform silk gland fibroin ADF-2 and ECP-1 (Guerette et al., 1996; Hu et al., 2005) and the aciniform silk gland fibroin AcSp1 (Hayashi et al., 2004)

However, no cDNA or protein sequences have been reported for the aggregate (secreting sticky glue) or the piriform (constructing attachment disc) silks There might be more spider silk fibroin genes awaiting isolation and characterization

Within spider silk glands, the fibroin silk proteins are maintained in a highly concentrated liquid crystalline form (Vollrath, and Knight, 2001) Many changes occur during passage of the spinning dope through the gland and duct, leading to the final and irreversible formation of the silk fibers It has been reported that, when the silk protein progresses along the narrow duct and aligns as insoluble thread, water content decreases

(Kojic, et al., 2004), pH decreases and ion composition changes (Dicko, et al., 2004c; Dicko, et al, 2004d) It is also indicated that the spinning conditions might play a role on the mechanical properties of the fibers (Gosline, et al., 1999; Vollrath, et al., 2001)

However, very little molecular information related to this silk formation process is available

Currently, the isolation, characterization and analysis of important genes and proteins responsible for various physiological processes are well underway in a wide range of organisms However, in the case of spiders, there is a clear gap in our understanding of the

molecular events underlying the silk formation and silk spinning process in vivo

In brief, there are two assumptions for us to initiate current research: there are still novel silk genes awaiting for identification and the EST approach is the one suitable to be utilized on this issue

Firstly, as mentioned above, the possibility of novel silk gene discovery exists Also

it is important to notice that previously identified silk genes essentially originate from protein sequences, which may therefore make further gene identification difficult as it is unlikely to isolate a gene without its protein information Nevertheless, to purify individual proteins from silk is not so easy, especially, some proteins may comprise only small amount of the silk

On the other hand, an EST approach is potent fpr novel gene identification Though it

is suggested by some authors that there might be genomic DNA contaminants, an EST database, if large enough, will absolutely be of great help in gene expression profiling and novel cDNA sequence isolation

In order to identify the molecular events underlying silk fibre formation, we have created a sequence database from all seven types of silk glands of a tropical spider species,

Nephila antipodiana, to initiate a first step for the understanding of the mystery of spider

silks’ formation

The objectives in the present study are:

1 To construct a cDNA library sourcing from the seven types of silk glands from

4 Structural analysis of silk proteins;

In conclusion, we start this project by construction of a cDNA library from Nephila

antipodiana silk glands Then, more than 1000 randomly selected clones are sequenced and

analyzed by homology searching in public database A number of potential targets that

might be implicated in spider silk fiber formation and processing were identified Of these, one novel spider fibroin gene from the tubuliform silk gland, TuSp1, has been identified and characterized

Chapter 2 Literature review

2.1 Silks in the nature

Silk is a proteinaceous polymer secreted by specialized exocrine glands in several groups of arthropods Animals in the classes Insecta and Arachnida (Phylum Arthropoda) produce silks (Craig, 2003) There are over 30,000 described species of spider that use silk

In addition, silks are produced by most of the 113,000 species in the insect order

Lepidoptera, the moths and butterflies, and by members of several other insect orders

Millions of years of evolution have resulted in silk used for various purposes: to construct protective shelter; to provide structural support; to catch and wrap prey The main difference between spiders and the silk producing insects is that spiders produce silks in their entire lifetime while certain types of insects usually produce silk in a specific stage of their life span The silk producing organs and the types of silk from spiders and insects evolve independently

Spider silks attract the attention from industry, military, and even popular media because of their remarkable properties: the tenacity is slightly lower than that of Nylon while the elasticity is twice as high (Forlix, 1996) Not surprisingly, the strength and elasticity of spider silk make it a good candidate for a broad range of medical and industrial applications, such as wound closure system, extremely fine sutures for neurosurgery, artificial tendons and ligaments, specific ropes, bulletproof vests and lightweight body armor, and other materials which require elasticity and tensile strength

2.2 Spider silks

2.2.1 Spider silk production organs

Spider silks are the secretory products of the spinning glands (Forlix, 1996) The spinning glands of spiders are basically a sac with an excretory duct, leading to a specific spinneret Generally the silk glands comprise a thin layer of seceretory epithelium (Kovoor, 1987; Ecophysiology of spiders, p169) Silk protein is synthesized in the epithelial cells and secreted into the glandular lumen as small droplets The silk protein could be stored in the middle part of the gland and exuded through the duct The transition from fibroin into silk fiber is irreversible

There are at least six kinds of spider silk glands that could be differentiated morphologically and histologically: ampullate glands; aciniform gland; tubuliform (cylindriform) glands; flagelliform glands; piriform glands; and aggregate glands (Forlix, 1996) Logically, the simplest silk glands occur in the ancient species (which generally own only one gland type), while there are up to seven kinds of different silk glands identified in the highly developed orb weavers (Forlix, 1996)

Figure 2.1 summarizes the specialized silk glands and the amino acid compositions

of the secretory product from each gland of a typical Araneid orb weaver (adapted from Vollrath and Knight, 2001)

Figure 2.1 The seven specialized glands and their different amino acid compositions of a typical Araneid orb weaver Silk glands are paired The aciniform

glands make the silk for wrapping prey, the tubuliform (cylindriform) glands make the cover silk for the egg sac and the pyriform glands make the silk for attachments and for joining threads The major and minor ampullate glands are of similar shape; the major glands provide the silk for the spider’s dragline and the frame threads of its web while the minor ones provide threads that can be added to any structural thread and may be used for the radial (spoke) threads of the web The flagelliform glands provide the core of the threads of the capture spiral, while the threads’ coating is supplied by the aggregate glands This coating contains glycoproteins for stickiness, salts to act as bactericides and hygroscopic organic compounds that, in addition to being bactericidal, also attract atmospheric water to plasticize the core of the thread (Adapted from Vollrath and Knight, 2001)

2.2.2 Spinning process of silks

Recently, much attention has been put on the spinning process, through which the soluble highly concentrated fibroin dope converts to the insoluble fiber Figure 2.2 depicts the spider spinneret while silk threads are emerging Figure 2.3A is a diagram of the silk

spinning apparatus while Figure 2.3B is a photo of the spinning spinneret of the Nephila

clavipes major ampullate silk gland, showing its internal anatomy (J.M Palmer

http://hubcap.clemson.edu/~ellisom/biomimeticmaterials/pictures/slidepictures/abdomen.gif)

In this process, the emulsion formation and micellar structures from aqueous solutions of reconstituted silkworm silk fibroin was observed (Jin and Kaplan, 2003), which was deemed as a first step in the silk formation process to control water and protein-protein interactions These micelles subsequently aggregated into larger ‘globules’ and gel-like states when silk fibroin increased Finally morphological alignment and silk formation

of the fibroin protein was demonstrated The process was believed to mimic the behavior of

the native silk protein in vivo

Observations on the major ampullate gland of the spider Nephila edulis also indicate

that the cuticle of the gland’s duct may have the structure of an advanced hollow fiber dialysis membrane and is thought to facilitate a rapid removal of water and change in ionic composition involved in the spinning process (Vollrath, and Knight, 1999) The element composition and pH change progressively as the dragline silk dope (spinning solution) passes down the duct to form the thread Na+ and Cl- composition decreased, K+ and P and

S increased, and pH dropped (Knight and Vallrath, 2001) It is thus concluded that precise control of the ionic environment within the spider’s spinning duct may be important in forming a tough insoluble thread (Vollrath and Knight, 2001, Figure 2.4)

In general, when the silk protein progresses along the narrow duct and aligns as

insoluble thread, water content decreases (Kojic, et al., 2004), pH decreases and ion composition changes (Dicko, et al., 2004c; Dicko, et al, 2004d) along the spinning duct It

is also indicated that the spinning conditions might play a role on the mechanical properties

of the fibers (Gosline, et al., 1999, Vollrath, et al., 2001) However, very scant molecular

information is available for elucidating this sophisticated process Therefore, it is necessary

to identify the molecular basis which is underlying the complex molecular events of silk formation

Figure 2.2 Spider spinneret (silk secreted from piriform gland spigot, Spiny Back

Spider, Castercantha sp.) (SEM x3,740)

This image is copyright of Dennis Kunkel at www.DennisKunkel.com, downloaded from http://www.emc.maricopa.edu/faculty/farabee/BIOBK/95139b.jpg

Figure 2.3A Diagrammatic representation of spinneret (Foelix, 1996)

Figure 2.3B Single spider spinneret showing the internal anatomy (Palmer)

From:http://hubcap.clemson.edu/~ellisom/biomimeticmaterials/pictures/slidepictures/abdomen.gif

Figure 2.4 A spider’s dragline spinneret The top part shows original drawings of the

histology of the spinneret (lumen, L); the bottom part outlines its spinning function, which entails drawing the liquid crystalline dope solution produced in the gland through a tapering s-shaped duct, thereby converting it into an elastic thread (Adapted fromVollrath, Knight, 2001)

2.2.3 Mechanical properties of spider silks

As shown in Tale 2.1, spider silks have remarkable features such as the combination

of high strength and elasticity

The material stiffness is indicated by the modulus (the slope of the stress strain curve); the strength of the materials is the maximal stress achieved before breaking; while the energy to break, however, is to measure the degree that the materials extend before they

Energy to break (Jkg-1) Spider frame silk

the insoluble silk formation, The formed silks are of excellent mechanical properties However, the cellular and molecular basis underlying this transition is needed for further investigation

2.3 Spider silk genes

2.3.1 Identification of spider silk genes

Since 1990, a number of spider silk cDNAs/genes have been identified The first spider silk gene was reported by Xu and Lewis in 1990 (Xu, and Lewis, 1990) In this report, dragline silk from the major ampullate silk gland was purified and digested The digested silk peptide was applied for amino acid sequencing DNA probes according to the amino acid sequence were synthesized and used to screen a major ampullate silk gland cDNA library Consequently, the first repetitive sequence (spidroin 1) of the fibroin protein

from major ampullate silk of the spider Nephila clavipes was determined from a partial

cDNA fragment of 2.4 kb in length According to the authors, the repeating unit is a maximum of 34 amino acids long and is not rigidly conserved The repeat unit is composed

of three different segments: (i) a 6 amino acid segment that is conserved in sequence but has deletions of 3 or 6 amino acids in many of the repeats; (ii) a 13 amino acid segment dominated by a polyalanine sequence of 5-7 residues; (iii) a 15 amino acid, highly conserved segment The latter is predominantly a Gly-Gly-Xaa repeat with Xaa being alanine, tyrosine, leucine, or glutamine The codon usage for this DNA is highly selective, avoiding the use of cytosine or guanine in the third position

Using similar strategies, a second partial cDNA clone for another dragline silk protein (Spidroin 2) was isolated by Hinman and Lewis (Hinman, and Lewis, 1992) in

1992, demonstrating that dragline silk is composed of multiple proteins The amino acid sequence exhibits an entirely different repetitive motif than Spidroin 1

In 1994, Beckwitt and Arcidiacono (1994) used the polymerase chain reaction (PCR)

to amplify the C-terminal part of Spidroin 1 gene from the spider Nephila clavipes Along

with some substitution mutations of minor consequence, the PCR-derived sequence reveals

an additional base missing from the previously published Nephila Spidroin 1 sequence Comparison of the PCR-derived sequence with the equivalent region of Spidroin 2 indicates that the insertion of this single base results in greatly increased similarity in the resulting amino acid sequences of Spidroin 1 and Spidroin 2 (75% over 97 amino acids)

The same PCR primers also amplified a fragment of the same length from Araneus

bicentenarius This sequence is also very similar to Spidroin 1 of Nephila (71% over 238

bases excluding the PCR primers, which translates into 76% over 79 amino acids) Therefore, it has been disclosed for the first time that there exists C-terminal conservation

of spider fibroin genes

The spider silk genes from another type of silk gland, the minor ampullate silk gland, are identified by Colgin,M.A and Lewis in 1998 (Colgin and Lewis, 1998) Two silk genes

from Nephila clavipes, MiSp 1 and 2, whose transcripts are 9.5 and 7.5 kb, respectively,

were determined by Northern blots Both MiSp proteins are organized into a predominantly repetitive region and a small nonrepetitive carboxy terminal region These highly repetitive regions are composed mainly of glycine and alanine, but also contain tyrosine, glutamine,

and arginine The sequences are mainly GGX and GA repeats The repetitive regions are interrupted by nonrepetitive serine-rich spacer regions Although the sequences of the spacer regions differ from the repetitive regions, sequences of the spacers from different regions of the proteins are nearly identical It is expected that the sequence differences between major and minor ampullate silks may explain the differing mechanical properties

of the fibers

In 1998, Hayashi, C Y et al., reported the cloning of Flag (Hayashi and Lewis,

1998), the substantial cDNA for flagelliform gland silk protein, which forms the core fiber

of the catching spiral Like all silks, the flagelliform protein is composed largely of iterated sequences The dominant repeat of this protein is Gly-Pro-Gly-Gly-X, which can appear up

to 63 times in tandem arrays This motif likely forms Pro2-Gly3 type II beta-turns and the resulting series of concatenated beta-turns are thought to form a beta-spiral It is therefore proposed that this spring-like helix is the basis for the elasticity of flagelliform silk The variable fifth position of the motif (X) is occupied by a small subset of residues (Ala, Ser, Tyr, Val) Moreover, these X amino acids occur in specific patterns throughout the repeats This ordered variation may suggest that with hydration, the beta-spirals form hydrogen-bonded networks that increase the elasticity of flagelliform silk

In addition, further sequencing analysis has shown that the exons and introns of the flagelliform gene underwent intragenic concerted evolution The intron sequences are more homogenized within a species than are the exons (Hayashi and Lewis, 2000) This pattern can be explained by extreme mutation and recombination pressures on the internally

repetitive exons The iterated sequences within exons encode protein structures that are critical to the function of silks Therefore, attributes that make silks exceptional biomaterials may also hinder the fixation of optimally adapted protein sequences

In 2004, a cDNA libraries derived from aciniform glands of the banded garden spider,

Argiope trifasciata, were constructed, and unique silk transcripts were sequenced (Hayashi,

et al., 2004) The inferred aciniform fibroin protein, aciniform spidroin 1 (AcSp1), is

composed of highly homogenized repeats that are 200 amino acids in length The long stretches of poly-alanine and glycine-alanine subrepeats, which are thought to account for the crystalline regions of minor ampullate and major ampullate fibers, are very poorly represented in AcSp1 The AcSp1 repeat unit is iterated minimally 14 times and does not display substantial sequence similarity to any previously described genes or proteins

Database searches showed that the nonrepetitive carboxy-terminus contains stretches

of matches to known spider fibroin sequences, suggesting that the AcSp1 gene is a highly divergent member of the spider silk gene family In phylogenetic analyses of carboxy-terminal sequences from araneid spiders, the aciniform sequence did not group strongly with clusters of fibroins from the flagelliform, minor ampullate, or major ampullate silk glands These data suggeste a possible linkage between silk fibroin sequences and performance of the different types of silks

In 1996, gland-specific expression of the spider silk gene, which allows for a range of mechanical properties according to the crystal-forming potential of the constituent fibroins,

was studied (Guerette, et al.,1996) One partial cDNA from the tubuliform silk gland

(ADF-2) was identified in this study However, the deduced amino acid composition of this protein is different from that of the silk from tubuliform silk gland, it is thus believed that there should be more genes to be identified as the major component from this gland

In 2005, another full length silk cDNA (ECP-1) tubuliform silk gland of Latrodectus

hesperus through a new strategy (Hu, et al., 2005a) In this study, the authors used the

strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, then isolated

an abundant component of spider egg case silk that is a 100-kDa protein doublet Combining mass spectrometry and reverse genetics, they have isolated a novel gene called ECP-1, which encodes for one component of the 100-kDa species BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-

1 These regions contained the conserved repetitive fibroin motifs poly(Ala) and Ala), however, no larger ensemble repeats could be identified within the primary sequence

poly(Gly-of ECP-1 Consistent with silk gland-restricted patterns poly(Gly-of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region In conclusion, this newly identified gene is much smaller than the traditional members of the spider silk gene family and lacks

of the C-terminal conservative region

In addition, to expand the spider silk gene database, sevencDNA libraries of silk

glands from five spider genera were made (Gatesy, et al., 2001) and partial cDNA or gene

sequences for 23 fibroins fromsix families of Araneae were obtained Conclusively, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumablyby stabilizing selection, over 125 million years of evolutionary history The retention of these conserved motifs since the Mesozoicand their convergent evolution in other structural superproteinsimply that these sequences are central for understanding the exceptionalmechanical properties of orb weaver silks

Briefly, araneoid spiders employ up to seven types of abdominal glands to produce silks for various purposes Since 1990, two silk genes MaSp1 and MaSp2 from the major ampullate silk gland (making dragline and frame silk, Xu, and Lewis, 1990; Hinman and

Lewis, 1992; Beckwitt and Arcidiacono, 1994; Guerette, et al., 1996; Beckwitt, et al., 1998; Gatesy, et al., 2001; Tian, et al., 2004), two silk genes from the minor ampullate silk gland (creating temporary capture spiral silk; Colgin and Lewis, 1998; Guerette, et al., 1996),

one from the flagelliform silk gland (producing core fiber of the spiral; Hayashi and Lewis, 1998; Hayashi and Lewis, 2000), one from the the aciniform silk gland (manufacturing

wrapping silk; Hayashi, et al., 2004) And two from the tubuliform silk gland (expressing egg sac silk; Guerette, et al., 1996; Hu, et al., 2005) have been identified Up to now, no

cDNA or protein sequences have been reported for the aggregate silk (secreting sticky glue)

or the piriform silk (constructing attachment disc)

Generally, the strategy for isolation and identification of novel silk genes is to screen

the gland-specific cDNA libraries using short degenerate probes based on known amino

acid sequences of silks from these specific glands This strategy has been demonstrated to

be very successful (Xu and Lewis, 1990; Hinman and Lewis, 1992; Colgin and Lewis,

1998; Hayashi and Lewis, 1998; Hayashi, et al., 2004) However, due to the deficiency of

sequence information of various silks, there are difficulties for designing new probes,

which may therefore hamper further discovery of novel silk cDNAs Another method is to

purify silk protein component, following by mass spectrometry and reverse genetics to

isolate novel silk genes (Hu, et al., 2005)

Table 2.2 Summarizes the information of the identified silk genes (modified from

Hayashi, 2002)

M37137 Nep.cla MaSp1 (Spidroin 1) Xu and lewis, 1990

1994 U37520 Nep.cla MaSp1 Beckwitt et al., 1998

U20329 Nep.cla MaSp1 Beckwitt et al., 1998

M92913 Nep.cla MaSp2 Hinman and Lewis, 1992

U20328 Ara.bic MaSp2 Beckwitt et al., 1998

1994 AF027735 Nep.cla MiSp1 Colgin and Lewis, 1998

AF027736 Nep.cla MiSp2 Colgin and Lewis, 1998

AF027737 Nep.cla MiSp2 Colgin and Lewis, 1998

AF027972 Nep.cla Flag Hayashi and Lewis, 1998

AF027973 Nep.cla Flag Hayashi and Lewis, 1998

AF218621 Nep.cla Flag Hayashi and Lewis, 2000

AF218622 Nep.cla Flag Hayashi and Lewis, 2000

AF218623 Nep.mad Flag Hayashi and Lewis, 2000

AF218624 Nep.mad Flag Hayashi and Lewis, 2000

U47853 Ara.dia ADF-1 (=MiSp) Guerette et al., 1996

U47854 Ara.dia ADF-2 Guerette et al., 1996

U47855 Ara.dia ADF-3 (=MaSp2) Guerette et al., 1996

U47856 Ara.dia ADF-4 Guerette et al., 1996

AF350262 Arg Aur MaSp1 Gatesy et al., 2001

AF350263 Arg Aur MaSp2 Gatesy et al., 2001

AF350264 Arg tri Flag Gatesy et al., 2001

AF350265 Arg tri Flag Gatesy et al., 2001

AF350266 Arg tri MaSp1 Gatesy et al., 2001

AF350267 Arg tri MaSp2 Gatesy et al., 2001

AF350268 Arg tri MaSp2-like Gatesy et al., 2001

AF350272 Gas Mam MASp2 Gatesy et al., 2001

AF350273 Lat Geo MaSp1 Gatesy et al., 2001

AF350274 Lat Geo MaSp2 Gatesy et al., 2001

SF350275 Lat Geo MaSp2 Gatesy et al., 2001

AF350276 Nep mad MaSp2-like Gatesy et al., 2001

AF350277 Nep mad MaSp1 Gatesy et al., 2001

AF350278 Nep mad MaSp2 Gatesy et al., 2001

AF350279 Nep sen MaSp1 Gatesy et al., 2001

AF350280 Nep sen MaSp2 Gatesy et al., 2001

AF350285 Tet kau MaSp1 Gatesy et al., 2001

AF350286 Tet ver MaSp1 Gatesy et al., 2001

AF350269 Dlo Ten Fibroin-1 Gatesy et al., 2001

AF350270 Dlo Ten Fibroin-2 Gatesy et al., 2001

AF350271 Eua Chi fibroin-1 Gatesy et al., 2001

AF350281 Ple tri Fibroin-1 Gatesy et al., 2001

AF350282 Ple tri Fibroin-2 Gatesy et al., 2001

AF350283 Ple tri Fibroin-3 Gatesy et al., 2001

AF350284 Ple tri Fibroin-4 Gatesy et al., 2001

AY571307 Kau.hib major ampullate spidroin 3 Tian et al., 2004

AY571308 Kau.hib major ampullate spidroin 1 Tian et al., 2004

AY571309 Kau.hib major ampullate spidroin 2-1 Tian et al., 2004

AY571310 Kau.hib major ampullate spidroin 2-2 Tian et al., 2004

AY566305 Age ape major ampullate spidroin Tian et al., 2004

AY426339 Arg tri AcSp1 Hayashi et al., 2004

AY994149 Lat hes ECP-1 Hu et al., 2005

2.3.2 Structure and organization of spider silk genes

2.3.2.1 Spider silk genes are large transcripts

In general, the spider silk genes are huge transcripts and the molecular size of their mRNA

exceeds 4 kb (Hayashi, et al., 1999) Tale 2.3 summarizes the information of the size of the

typical silk fibroin genes from major ampullate, minor ampullate, flagelliform and aciniform silk gland

Table 2.3 Summarizes of the size of the the typical silk fibroin genes (Modified

from Hayashi, et al., 1999)

origin of silk protein cDNA Length (kb)

2.3.2.2 Spider silk genes contain internal repetitive sequences

From the beginning of spider silk gene identification, it has been noticed that there exists internal repetitive sequences For example, Figure 2.5 shows amino acid sequence of MaSp2 (Hinman, M.B and Lewis, R.V, 1992), which were rearranged to show the internal repetitive elements

Figure 2.5 Predicted amino acid sequence for the Spidroin 2 protein, rearranged to show repetitive elements The most repetitive protein elements have been arranged from

the amino-terminal though the highly conserved region, followed by the less conserved region and divergent COOH-terminal tail The dashes represent deletions, allowing the elements to be arranged for maximum identity (Hinman and Lewis, 1992)

There are attempts to try to generalize these internal repetitive sequences into simple consensus repetitive elements (Hayashi, 2002), which will be able to be used for representation and characterization of the silk proteins However, as the spider silk gene database expanded, it has been found that this simplification is not easy, as the repetitive elements of silk proteins from different sources are quite diversified

Figure 2.6 depicted some of the consensus repeats in the typical silk genes from N

clavipes (Hayashi, et al., 1999)

Figure 2.6 The silk proteins from N clavipes are depicted as generalized, or

consensus, amino acid repeats Subscripts indicate the number of times a sequence

is tandemly repeated (Hayashi, C.Y., et al., 1999)

As shown above, the length and amino acid composition of the repetitive units from each member of the silk gene family are different On the other hand, the silks from difference glands are of different mechanical properties and functions It is natural to try to connect the sequence information with the mechanical properties Various combinations of these simple motifs are suggested to form the structural modules of silk fibers from various types of silk glands, and to be critical for determining the mechanical attributes of the silk

2.3.2.3 Spider silk genes have a C-terminal conservative region

As mentioned above (Beckwitt and Arcidiacono, 1994), through the PCR-derived sequence analysis, it has been revealed an additional base missing from the previously

published Nephila Spidroin 1 sequence It is therefore disclosed for the first time of the

C-terminal conservation of spider fibroin genes

When the number of identified silk genes increased, the C-terminal conservation was observed of the fibroin genes from major ampullate, minor ampullate, flagelliform, and aciniform silk gland (Beckwitt and Arcidiacono, 1994; Colgin and Lewis, 1998; Hayashi

and Lewis, 1998; Hayashi, et al., 2004) The high sequence conservation in the

carboxy-terminal region is thus used for novel silk gene identification and to infer the evolutionary relationship among the fibroin gene family members

An immunological method was applied recently to demonstrate the existence of the conserved C-terminal part in the ampullate spinning dope and the mature silk thread

(Sponner, et al., 2004) Polyclonal antibodies derived against fusion proteins containing the