Plasticity of cardiac titin/connectin in heart development

Many sarcomeric proteins in the myocardium alter their isoform pattern during perinatal development to adjust to the intensified pump function of the postnatal heart. These changes also involve the giant protein titin/connectin. Here we show by low-percentage polyacrylamide-gel electrophoresis that developmentally regulated switching of cardiac titin/connectin size occurs in the hearts of mouse, rat, pig, and chicken. Mammalian hearts express, well before birth, large foetal (∼3.7 MDa) N2BA-titin/connectin isoform but no N2B-isoform (3.0 MDa). During perinatal heart development the 3.7-MDa N2BA-isoform is replaced by a mix of smaller isoforms. At birth a plethora of intermediate-size N2BA-isoforms appears together with the N2B-isoform. In postnatal heart development the larger-size N2BA-isoforms disappear and smaller-size N2BA-isoforms are upregulated, whereas the proportion of N2B-titin/connectin increases to species-specific adult levels. The time courses of isoform switching are faster in small than in large mammals. Titin/connectin isoform switching also takes place in developing chicken hearts, but the largest embryonic isoform found here was less than 3.4 MDa. At hatching, various smaller-size isoforms appeared and within a week the adult expression pattern was established representing a major 3.0-MDa isoform and a minor 3.15-MDa isoform. The ratio between the two adult isoforms differed between the left ventricle and the right atrium. The perinatal changes toward smaller cardiac titin/connectin isoforms in mammals and chicken greatly increase the myofibrillar passive tension of postnatal hearts. Plasticity of titin/connectin at approximately the time of birth thus affects myocardial mechanics but could also be an important factor in developmentally regulated assembly and signalling processes.Proceedings of the International Symposium on Muscle Elastic Proteins:Koscak Maruyama Memorial Meeting, Chiba, Japan, November 2004     

Calcium binding to an elastic portion of connectin/titin filaments

-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the -connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on -connectin. Purified -connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 10⁷ M^–1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 M. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of -connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of -connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl₂ and 70 M leupeptin to split connectin filaments into -connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while - and -connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 m at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N₂ line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction–relaxation cycle of skeletal muscle.     

Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of “idiopathic” cardiomyopathy. Recent molecular genetic analyses have now revealed that “idiopathic” cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.     

The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study

Summary Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 m, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 m, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.     

Behaviour of connectin (titin) and nebulin in skinned muscle fibres released after extreme stretch as revealed by immunoelectron microscopy

Summary Stretching of skinned fibres of frog skeletal muscle beyond the overlap of the thin and thick filaments followed by release to resting length results in disorganization of the thin filaments at the A-I junction of a sarcomere (Higuchiet al. (1988) J. Muscl. Res. Cell. Motility 9, 491–8). Immunoelectron microscopic observations showed that the binding sites of antibodies against connectin (titin) returned to the original position after extreme stretch and release but those of anti-nebulin antibodies were largely disorganized. The binding sites of anti-connectin antibodies moved within an I band with the change in sarcomere length, but those of anti-nebulin antibodies did not. Nebulin remained in the I band at extreme stretch. Thus connectin filaments appear to be responsible for maintaining mechanical continuity of a sarcomere and appear to behave independently of thin filaments. It is suggested that nebulin is localized in the I band but not in the A band and is associated with thin filaments but not with the elastic structure of myofibrils.     

Effects of eccentric exercise on joint stiffness and muscle connectin (titin) isoform in the rat hindlimb

We investigated the effects of repeated eccentric exercise for rat medial gastrocnemius muscle on ankle joint stiffness and muscle connectin (titin) isoform composition (longer form, alpha-connectin; shorter form, beta-connectin). Male Wistar rats were trained on a custom-made, isokinetic dynamometer (eccentric-exercise group, n = 6; sham-operated group, n = 6). The exercise session consisted of 20 eccentric contractions elicited by submaximal electric stimulations under anesthesia. The contracting muscle was forcibly lengthened by an isokinetic dorsi-flexion of the ankle joint (velocity, 30 degrees/s; range of motion, 45 degrees). Rats in the eccentric-exercise group were trained every two days for 20 days (10 sessions in total). The static passive resistive torque (PRT) of 45 degrees at the ankle joint was used as a measure of the joint stiffness, and was determined before and after the experimental period. After 10 sessions of eccentric exercise, the wet weight of medial gastrocnemius muscle significantly increased (P < 0.05), whereas the static PRT significantly decreased (P < 0.05) in the eccentric-exercise group, when compared to the sham-operated group. Myosin-ATPase staining showed a decrease in the number of type IIb/IId fibers (P < 0.001) and an increase in the number of type IIa fibers (P < 0.05). However, no significant difference was seen in the connectin (titin) isoform composition between the eccentric-exercise group and the sham-operated group, suggesting that the reduction in PRT was not due to change in resting mechanical properties of muscle fibers.     

Developmental changes in rat cardiac titin/connectin: transitions in normal animals and in mutants with a delayed pattern of isoform transition

Rat cardiac titin undergoes developmental changes in isoform expression during the period from late embryonic through the first 20–25 days of life. At least five size classes of titin isoforms have been identified using SDS agarose gel electrophoresis. The longest normal isoform is expressed in the embryonic stages, and it is progressively replaced with increasingly smaller versions. The isoform switching is consistent with changes in resting tension from lower values in one-day neonates to higher levels in adult myocytes. Considerable micro-heterogeneity in alternative splicing patterns also was found, particularly in the N2BA PEVK region of human, rat, and dog ventricle. A rat mutation has been identified in which the embryonic-neonatal titin isoform transitions are markedly delayed. These mutant animals may prove useful for examining the role of titin in stretch-activated signal transduction and in the Frank–Starling relationship.     

Assembly of the cardiac I-band region of titin/connectin: expression of the cardiac-specific regions and their structural relation to the elastic segments

Summary The giant molecule titin (also called connectin) provides an elastic connection in the I-band between the Z-disk and A-band of striated muscle. This region is assembled in a tissue-specific way by extensive differential splicing events. We have raised monoclonal antibodies against the two N2-line isoforms of titin and demonstrate that both forms of cardiac I-band titin are constitutively co-expressed in atrial and ventricular muscle. In developing mouse embryos, the expression of the cardiac N2-B isoform remains strictly cardiac-specific and is linked to the expression of the ubiquitous N2-A isoform. The mechanical function of the cardiac N2-line region was investigated ultrastructurally. Immunoelectron microscopy reveals that the N2-B region separates two mechanically distinct sections of titin with a hyperextensible segment spanning the distance to the Z-disk. The formation of a plateau in the extension of cardiac titin rules out that Ig-domains can be unfolded as a mechanism of elasticity.     

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments

Summary The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 m remained at about 0.8 m from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 m. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 m of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.     

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin

Summary A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3 end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.     

Projectin is an invertebrate connectin (titin): Isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle

Summary A filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass ( 1200 kDa) was considerably lower than that of connectin ( 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connectin reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein crossreacted with chicken connectin. Circular dichroic spectra indicated the abundance of-sheet structure ( 60 %). Low-angle shadowed images showed filamentous structures (0.2 0.5m) by electron microscopy. Proteolysis of the 1200 kDa protein by -chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connectins and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin.Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle.It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated minititin estimated as 600 kDa appears to be a proteolytic product ( 1100 kDa) of the parent molecule ( 1200 kDa).     

Maximal voluntary force and rate of force development in humans--importance of instruction

The present investigation has been designed to confirm the effect of instruction (hard-and-fast instruction compared to fast instruction) upon maximal voluntary isometric force (MVF) and maximal rate of force development (MRFD) in muscle groups which differ with regards to muscle mass and usage. In addition, we took advantage of the force data collected during unilateral and bilateral leg extension, to compare the instruction effects on the indices of the bilateral deficits (BI, the differences between the data collected during bilateral extensions and the sum of the data collected during unilateral left and right extensions) with regard to MVF (BI_MVF) and MRFD (BI_MRFD). Force-time curves were recorded during maximal isometric contractions of the elbow flexors, the leg extensors of the take-off and lead legs and during bilateral leg extension in 26 healthy young male volunteers from the track-and-field national team of Tunisia. In the first protocol, the subjects were instructed to produce MFV as hard-and-fast as possible (instruction I). In the second protocol (instruction II) the subjects were instructed to provide MFRD, that is the most explosive force, by concentrating on the fastest contraction without concern for achieving maximal force. The present study confirmed the importance of an appropriate instruction for the measurement of MRDF. The MRFD (F=40.8, P<0.001) were significantly higher when measured after instruction II compared to instruction I. The effect of the instruction upon MRFD were similar for muscle groups with different volumes, cortical representations and uses. The same results (F=52.1; P<0.001) were observed when MRFD was related to MVF [MRFD%=(MRFD/MVF)×100]. On the other hand, MVF was similar following both instructions (ANOVA, F=0.562; P=0.454). Moreover, the results of the present study suggested that the effect of instruction was significantly larger for BI_MRFD than forBI_MVF. Electronic Publication     

Non-invasive assessment of single motor unit mechanomyographic response and twitch force by spike-triggered averaging

A method for non-invasive assessment of single motor unit (MU) properties from electromyographic (EMG), mechanomyographic (MMG) and force signals is proposed. The method is based on the detection and classification of single MU action potentials from interference multichannel surface EMG signals and on the spike-triggered average of the MMG (detected by an accelerometer) and force signals. The first dorsal interosseous (FDI) and abductor digiti minimi (ADM) muscles were investigated at contraction levels of 2% and 5% of the maximum voluntary contraction (MVC) force. A third contraction was performed by selective activation of a single MU with surface MU action potential visual feedback provided to the subject. At 5% MVC, the mean (±standard error) single MU MMG peak-to-peak value was 11.0±1.8 mm s⁻² (N=17) and 32.3±6.5 mm s⁻² (N=20) for the FDI and AMD muscles, respectively. The peak of the twitch force was, at the same contraction livel, 7.41±1.34 mN and 14.42±2.92 mN, for the FDI and ADM muscles, respectively. The peak-to-peak value of the MMG was significantly different for the same MU at different contraction levels, indicating a non-linear summation of the single MU contributions. For the FDI muscle, the MMG peak-to-peak value of individual MUs was 21.5±7.8 mm s⁻², when such MUs were activated with visual feedback provided to the subject, whereas, for the same MUs, it was 11.8±3.8 mm s⁻², when the subject maintained a constant force level of 2% MVC. The method proposed allows the non-invasive assessment of single MU membrane and contractile properties during voluntary contractions.     

Poly-Ig tandems from I-band titin share extended domain arrangements irrespective of the distinct features of their modular constituents

The cellular function of the giant protein titin in striated muscle is a major focus of scientific attention. Particularly, its role in passive mechanics has been extensively investigated. In strong contrast, the structural details of this filament are very poorly understood. To date, only a handful of atomic models from single domain components have become available and data on poly-constructs are limited to scarce SAXS analyses. In this study, we examine the molecular parameters of poly-Ig tandems from I-band titin relevant to muscle elasticity. We revisit conservation patterns in domain and linker sequences of I-band modules and interpret these in the light of available atomic structures of Ig domains from muscle proteins. The emphasis is placed on features expected to affect inter-domain arrangements. We examine the overall conformation of a 6Ig fragment, I65–I70, from the skeletal I-band of soleus titin using SAXS and electron microscopy approaches. The possible effect of highly conserved glutamate groups at the linkers as well as the ionic strength of the medium on the overall molecular parameters of this sample is investigated. Our findings indicate that poly-Ig tandems from I-band titin tend to adopt extended arrangements with low or moderate intrinsic flexibility, independently of the specific features of linkers or component Ig domains across constitutively- and differentially-expressed tandems. Linkers do not appear to operate as free hinges so that lateral association of Ig domains must occur infrequently in samples in solution, even that inter-domain sequences of 4–5 residues length would well accommodate such geometry. It can be expected that this principle is generally applicable to all Ig-tandems from I-band titin.     

人血小板/T细胞活化抗原1(PTA1)亲和层析柱的制备及PTA1/Ig的纯化

目的：制备可用于纯化血小板／Ｔ细胞活化抗原１（ｐｌａｔｅｌｅｔａｎｄＴｃｅｌａｃｔｉｖａｔｉｏｎａｎｔｉｇｅｎ１，ＰＴＡ１）融合蛋白的亲和层析柱，纯化真核表达的ＰＴＡ１／Ｉｇ融合蛋白，以进一步研究ＰＴＡ１的功能及其配体。方法：以硫酸铵及阴离子交换色谱法纯化抗ＰＴＡ１ｍＡｂ，并交联Ｓｅｐｈａｒｏｓｅ４Ｂ制备ＰＴＡ１亲和层析柱。通过亲和层析法，从ｐＰＴＡ１／Ｉｇ表达载体转染的ＣＯＳ７细胞培养上清中纯化ＰＴＡ１／Ｉｇ融合蛋白，用夹心ＥＬＩＳＡ及ＳＤＳＰＡＧＥ鉴定纯化结果。结果：硫酸铵及阴离子交换色谱纯化后，获得纯度高和活性好的抗ＰＴＡ１ｍＡｂ，用其交联Ｓｅｐｈａｒｏｓｅ４Ｂ制备ＰＴＡ１亲和层析柱，交联率为９６％。该亲和层析柱可从ｐＰＴＡ１／Ｉｇ转染的ＣＯＳ７细胞上清每１００ｍｌ中，纯化ＰＴＡ１／Ｉｇ融合蛋白约１２６μｇ。结论：制备成功可用于纯化ＰＴＡ１的亲和层析柱，并获得纯化的ＰＴＡ１／Ｉｇ融合蛋白，为进一步研究ＰＴＡ１的功能及鉴定其配体提供了有力手段     

Adapted protocol of rate of force development and relaxation scaling factor for neuromuscular assessment in patients with knee osteoarthritis

BackgroundThe linear relationship between muscle torque and rate of torque rise/relaxation during rapid muscle contractions has been recently introduced as a novel measure of muscle quickness, termed rate of torque development/relaxation scaling factor (RTD-SF/RTR-SF). Because the standard assessment protocol includes potentially painful muscle contractions, the first purpose of this study was to validate an adapted RTD-SF/RTR-SF protocol for knee extensor muscles that utilizes lower submaximal intensities and can be used in knee osteoarthritis patients.MethodsA cross-sectional study was performed on a group of healthy controls (n = 24) who underwent the standard RTD-SF/RTR-SF protocol (20–80% of maximum) and the knee osteoarthritis group (n = 24) who underwent the adapted protocol (20–60% of maximum). We calculated the RTD-SF, RTR-SF and the linearity (r²) for both relationships, based on both protocols in controls.ResultsThe validity of the adapted protocol was acceptable (intraclass correlation coefficient = 0.77–0.93), with low within-participant variation (coefficient of variability <10%) for both outcome measures. Compared with the control group, the knee osteoarthritis group had similar RTD-SF, but lower linearity of RTD-SF (0.90 vs. 0.82). The RTR-SF (8.0/s vs. 6.7/s) and its linearity (0.87 vs. 0.73) were significantly reduced. Comparing the affected and the unaffected leg in the knee osteoarthritis group, the unaffected leg had greater maximal torque (96.2 vs. 84.1 Nm) and higher linearity for RTD-SF (0.86 vs. 0.80) and RTR-SF (0.82 vs. 0.73).ConclusionsWe confirmed the validity of the adapted RTD/RTR-SF protocol and its sensitivity to impairments associated with knee osteoarthritis.     

Unilateral arm strength training improves contralateral peak force and rate of force development

Neural adaptation following maximal strength training improves the ability to rapidly develop force. Unilateral strength training also leads to contralateral strength improvement, due to cross-over effects. However, adaptations in the rate of force development and peak force in the contralateral untrained arm after one-arm training have not been determined. Therefore, we aimed to detect contralateral effects of unilateral maximal strength training on rate of force development and peak force. Ten adult females enrolled in a 2-month strength training program focusing of maximal mobilization of force against near-maximal load in one arm, by attempting to move the given load as fast as possible. The other arm remained untrained. The training program did not induce any observable hypertrophy of any arms, as measured by anthropometry. Nevertheless, rate of force development improved in the trained arm during contractions against both submaximal and maximal loads by 40-60%. The untrained arm also improved rate of force development by the same magnitude. Peak force only improved during a maximal isometric contraction by 37% in the trained arm and 35% in the untrained arm. One repetition maximum improved by 79% in the trained arm and 9% in the untrained arm. Therefore, one-arm maximal strength training focusing on maximal mobilization of force increased rapid force development and one repetition maximal strength in the contralateral untrained arm. This suggests an increased central drive that also crosses over to the contralateral side.     

Sequence analysis and antigen binding characteristics of Ig SCID Ig+ mice

SCID mice as they age may develop a limited number of functionalB and T cell clones. V_H and V_L gene sequences of hybridoma antibodiesderived from the spleens of five adult SCID Ig⁺ mice revealednine clonally unrelated, i.e. derived from different precursorlymphocytes, groups of Ig. Of these, four used the V_H7183 family,one Q52, three J558 and one V_H3609. V_H7183 was seen in excessover J558 despite a 5-fold larger number of J558 family membersin the mouse genome. Preferential use of D proximal V^Hgenesalong with reduced N region addition in the heavy chain VDJjoins of these antibodies suggest derivation from a populationof lymphocytes with characteristics of the perinatal repertoire.All of the hybridomas produced light chains of several differentV_L family groups. Sequences of 23 V_H and 22 V_L genes of hybridomasderived from one mouse (no. 45) showed that they were all clonallyrelated, differing only in somatic mutations. Autoreactivityof these antibodies to cellular antigens [including (U1)RNP-specificA protein and SmB] was analyzed, and shown to correlate withthe geneology and specific mutations within members of the clone.The geneological tree derived from sequence data permitted astudy of the microevolution of a clone of B cells and showedthat as it developed there was a tendency toward increased strengthof self-reactive binding as well as alterations in specificitytoward these autoantigens. Analysis of replacement: silent mutationratios within members of this clone reflected limited affinitymaturation in mouse 45 and none for another mouse, no. 58. Thesedata are consistent with the progressive differentiation ofB cells in an immunodeficient state in the absence of clonalcompetition with B cells of similar antigen specificities asthey populate the spleen.