Alternative mRNA Processing Research Articles

Yeast CBP1 mRNA 3' end formation is regulated during the induction of mitochondrial function.

Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene. While many mammalian genes contain multiple mRNA 3' cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity. The Saccharomyces cerevisiae nuclear gene CPB1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability. This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CPB1. Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3' end near the middle of the coding sequence. Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA. Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3' end formation of the 1.2-kb transcript, a unique case in yeast cells.

A human integrin β1 subunit with a unique cytoplasmic domain generated by alternative mRNA processing

The integrin subunit (β1) is common to a group of plasma membrane glycoprotein heterodimers that include the fibronectin, laminin and collagen receptors. These receptors span the plasma membrane, providing a transmembrane linkage between the extracellular matrix and the cytoskeleton. Here, we describe a variant of the human β1 differing from the previously described β1 in the cytoplasmic domain. The variant β1 transcript (β1 3′v is present in different cell types and is synthesized at lower levels compared to the β1 mRNA. The cytoplasmic domain of the β1 3′v is characterized by a unique 12-amino acid C-terminal sequence. A Tyr residue present in this region, and known to be phosphorylated in the β1, is no longer part of a consensus sequence for phosphorylation by Tyr kinases. The integrin cytoplasmic domain anchors actin fibrils to the plasma membrane by interacting with cytoskeletal proteins such as talin and fibulin. The integrin β1 3′v with the variant cytoplasmic domain is likely to mediate a new type of membrane-cytoskeleton interaction during cell-cell and cell-matrix adhesion. Analysis of genomic clones showed that the new sequences of the variant mRNA are identical to an intron located between the last two exons of the β1 gene, indicating that the alternative message is generated either by premature transcription termination or by lack of splicing at this site.

Distinct vascular and intestinal smooth muscle myosin heavy chain mRNAs are encoded by a single-copy gene in the chicken

We examined whether the gizzard MHC gene is expressed in other smooth muscle tissues and, if so, whether there exist any smooth muscle MHC isoforms at the mRNA level. Northern blot analysis showed that the gizzard MHC gene was also expressed in the aorta and jejunum, but not in the pectoralis muscle or in fibroblasts. This indicates that striated muscle and non-muscle MHC isoforms are encoded in genes distinct from the smooth muscle MHC gene. Further, nuclease S1 mapping showed that the aortic smooth muscle MHC mRNA was distinct from the gizzard mRNA in the 5′-terminal coding region. Both of these mRNA species are expressed in the jejunum. These observations suggest that there exist at least two chicken smooth muscle MHC isoforms, vascular-type and intestinal-type, and that these isoforms are generated from a single-copy gene, probably by an alternative mRNA processing mechanism.

Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.

Translational Control of Germ Cell-Expressed mRNA Imposed by Alternative Splicing: Opioid Peptide Gene Expression in Rat Testis

The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.

Alternative usage of different poly(A) addition signals for two major species of mRNA encoding human aromatase P-450

Two cDNA clones for human placental aromatase P-450 (P-450 AROM) have been isolated and sequenced. The insert of one clone (2894 bp) contains an open reading frame encoding a protein consisting of 503 amino acid residues together with a 49 bp 5′-untranslated stretch and a 1336 bp 3′-noncoding region to which a poly(A) tract is attached. Three potential poly(A) addition signals are detected in this 3′-noncoding region. The other clone contains a shorter cDNA insert, the nucleotide sequence of which overlaps with most of the sequence of the longer cDNA insert (nucleotides 36–2355) except for one nucleotide substitution. The 3′-noncoding region of this shorter cDNA is only 846 bp in length, but a poly(A) tract is also attached to its 3′-terminus. Northern blot analysis of human placental RNA reveals the presence of two major mRNA species of 3.4 and 2.9 kb when probes excised from the overlapping region of these two cDNAs are employed. The 2.9 kb mRNA is not detected, however, when a fragment of the non-overlapping region of the longer cDNA is used as a probe. It is therefore concluded that the two major species of P-450 AROM mRNA are formed as a consequence of alternative processing of precursor mRNA(s).

Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences.

In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.

Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences.

In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.

Analysis of Mammalian Pigmentation at the Molecular Level

There has been great interest lately in the cloning of pigment-related genes; several laboratories have succeeded in isolating melanocyte-specific genes which have many of the characteristics expected for tyrosinase. In this paper, we review the selection criteria, the physical properties, and the functional characteristics of several of these gene products. Two of the clones map to the brown (b) and albino (c) loci, genes that are involved in the regulation of the quantity and quality of melanin production. The functional characteristics of these gene products are not easily reconciled with existing schemes of melanogenesis, and a reevaluation of our concepts of melanogenic regulation may be necessary. The altered expression of these gene products in normal and in transformed melanocytes, and the alternative mRNA processing that occurs in those cells, makes this system an appropriate and interesting one for studies of normal metabolic regulation of gene expression, as well as altered gene expression by neoplastic cells.

Substance P regulates the vasodilator activity of calcitonin gene-related peptide

The 37-amino-acid calcitonin gene-related peptide (CGRP) occurs as a result of alternative processing of mRNA from the calcitonin gene. The potency of CGRP as a vasodilator and the occurrence of the peptide in nerves associated with blood vessels suggest an important role for CGRP in the regulation of blood flow. The finding that CGRP induces protracted vasodilatation when administered extra-vascularly, to mimic release from nerves, has led us to investigate how the vasodilator activity of CGRP is controlled in vivo. CGRP is often co-localized with substance P in C-fibre nerves. Here, we demonstrate that injection of CGRP with substance P into human skin converts the long-lasting vasodilatation induced by CGRP into a transient response. Experiments in animals reveal that the phenomenon is dependent on the action of proteases from mast cells stimulated by substance P. The results reveal a new regulatory interaction between two neuropeptides and provide evidence for an in vivo role for mast cell proteases.

The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing.

Tropomyosin (TM), a ubiquitous protein, is a component of the contractile apparatus of all cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and nonsarcomeric muscle, it is a component of the thin filament. Several different TM isoforms specific for nonmuscle cells and different types of muscle cell have been described. As for other contractile proteins, it was assumed that smooth, striated, and nonmuscle isoforms were each encoded by different sets of genes. Through the use of S1 nuclease mapping, RNA blots, and 5' extension analyses, we showed that the rat alpha-TM gene, whose expression was until now considered to be restricted to muscle cells, generates many different tissue-specific isoforms. The promoter of the gene appears to be very similar to other housekeeping promoters in both its pattern of utilization, being active in most cell types, and its lack of any canonical sequence elements. The rat alpha-TM gene is split into at least 13 exons, 7 of which are alternatively spliced in a tissue-specific manner. This gene arrangement, which also includes two different 3' ends, generates a minimum of six different mRNAs each with the capacity to code for a different protein. These distinct TM isoforms are expressed specifically in nonmuscle and smooth and striated (cardiac and skeletal) muscle cells. The tissue-specific expression and developmental regulation of these isoforms is, therefore, produced by alternative mRNA processing. Moreover, structural and sequence comparisons among TM genes from different phyla suggest that alternative splicing is evolutionarily a very old event that played an important role in gene evolution and might have appeared concomitantly with or even before constitutive splicing.

Structure-function studies of the amino-terminal region of bovine cardiac troponin T.

The length and amino acid sequence of the amino-terminal region of troponin T (TnT) is regulated by alternative mRNA processing in both mammals and birds. To study the function of this region, three forms of bovine cardiac TnT were compared: isoforms TnT1 and TnT2, which differ by the presence or absence of residues 15-19 and TnT 39-284. TnT 39-284 was prepared by chemical cleavage of TnT1 at Cys-39. All three forms of TnT successfully reconstituted with troponin I and troponin C, resulting in troponins designated Tn1, Tn2, and TnCN. Three properties of the reconstituted troponins were compared. 1) Tn1 and TnCN had indistinguishable effects on tropomyosin polymerization. Addition of either 8 microM Tn1 or 8 microM TnCN increased the viscosity (eta rel) of 5 microM tropomyosin from 1.0 to 1.63 at 10 degrees C. 2) All of the three troponins conferred Ca2+ dependence to the MgATPase rate of myosin S-1-actin-tropomyosin. In the presence of saturating concentrations of Tn2, Tn1, or TnCN, 50% MgATPase activation occurred at pCa 6.0, 5.9, or 5.75, respectively. 3) The affinity of the Ca2+-specific binding site of reconstituted Tn1 was 50% stronger than the affinity of the same site on TnCN. These results suggest that the amino-terminal region of cardiac TnT is not a completely Ca2+-insensitive domain, but rather modulates the interaction of Ca2+ with troponin and with the thin filament. Furthermore, the effects of TnT on tropomyosin-tropomyosin binding are predominantly due to portions of TnT carboxyl-terminal to residue 38.

Divergence in primary structure between the molecular forms of acetylcholinesterase.

We have isolated a COOH-terminal tryptic peptide from the hydrophobic globular (5.6 S) form of Torpedo californica acetylcholinesterase that exhibits divergence in amino acid sequence from the catalytic subunit of the dimensionally asymmetric (17 S + 13 S) enzyme. The divergent peptide could be recovered from the glycophospholipid-modified 5.6 S enzyme only after treatment with phosphatidylinositol-specific phospholipase C. Upon reduction, carboxymethylation with [14C]iodoacetate, and trypsin digestion the resultant peptides were purified by gel filtration followed by high performance liquid chromatography. The high performance liquid chromatography profiles of 14C-labeled cysteine peptides from lipase-treated 5.6 S enzyme revealed unique radioactive peaks which had not been present in digests of the asymmetric form. These peaks all yielded identical amino acid sequences. The difference in chromatographic behavior of the individual peptides most likely reflects heterogeneity in post-translational processing. Gas-phase sequencing and composition analysis are consistent with the sequence: Leu-Leu-Asn-Ala-Thr-Ala-Cys. Composition includes 2-3 mol each of glucosamine and ethanolamine which is indicative of modification by glycophospholipid. Glucosamine is also present in an asparagine-linked oligosaccharide. The two forms of acetylcholinesterase diverge after the threonine residue within this peptide sequence; the hydrophobic form terminates with cysteine whereas the asymmetric form extends for 40 residues beyond the divergence. The locus of divergence and absence of any other amino acid sequence difference suggest that the molecular forms of acetylcholinesterase arise from a single gene by alternative mRNA processing.

Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing.

We analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs there is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing.

We analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs there is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures.

Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1. The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region. Species with 5' ends in E7 accounted for over 95% of all transcripts seen. Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region. The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region. This species probably encodes the abundant E4 protein found in plantar warts (F. Breitburd, O. Croissant, and G. Orth, Cancer Cells, vol. 5, in press; J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986). Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF. The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Calcitonin gene-related peptide is localised to human airway nerves and potently constricts human airway smooth muscle.

In human airways synthetic human sequence calcitonin gene-related peptide (hCGRP), a novel peptide produced by alternative processing of mRNA from the calcitonin gene, caused concentration-dependent contraction of human bronchi (EC50 4.9 X 10(-9) M) and was significantly more potent than substance P or carbachol. The contractile response was unaffected by atropine (2 X 10(-6) M), propranolol (10(-6) M), indomethacin (10(-5) M), tetrodotoxin (3 X 10(-6) M), chlorpheniramine (10(-4) M), cimetidine (10(-5) M), or FPL55712 (10(-4) M) suggesting a direct effect of CGRP on airways smooth muscle. CGRP was detected in human airways by radioimmunoassay with highest concentrations in cartilaginous airways. CGRP was localised by immunocytochemistry to both nerves and ganglia in human airways. CGRP, is a potent constrictor of human airways and may have important effects on airway function and be implicated in the pathogenesis of bronchial hyper-responsiveness and asthma.

Alternative processing of bovine growth hormone mRNA: nonsplicing of the final intron predicts a high molecular weight variant of bovine growth hormone.

We have detected a variant species of bovine growth hormone mRNA in bovine pituitary tissue and in a stably transfected bovine growth hormone-producing cell line. Analysis of this variant mRNA indicated that the last intervening sequence (intron D) had not been removed by splicing. Inspection of the sequence of intron D reveals an open reading frame through the entire intron, with a termination codon encountered 50 nucleotides into the fifth exon, which is shifted from the normal reading frame in this variant mRNA. If translated, this variant mRNA would encode a growth hormone-related polypeptide having 125 amino-terminal amino acids identical to wild-type growth hormone, followed by 108 carboxyl-terminal amino acids encoded by the 274 bases of intron D along with the first 50 nucleotides of exon 5. This variant polypeptide would be 42 amino acids longer than wild-type bovine growth hormone or approximately 5000 greater in molecular weight. The intron D-containing variant of bovine growth hormone mRNA was demonstrated to exist on polysomes, suggesting that this mRNA species is translated into a polypeptide. Cytosolic mRNA species containing any of the other three introns of the bovine growth hormone gene were not detectable.

Multiple purine pathway enzyme activities are encoded at a single genetic locus in Drosophila.

The Drosophila melanogaster Gart locus, known from previous work to encode the enzyme activity phosphoribosylglycinamide formyltransferase (GART), specifies two alternatively processed mRNAs and two proteins. We introduced the entire Gart locus into a Drosophila tissue culture cell line in which the locus is active. The resulting cell clones contained numerous copies of the locus and overproduced both mRNAs and both expected proteins, thus markedly facilitating analysis of these molecules. We assayed extracts of the clones for the activities of 10 enzymes important for de novo purine synthesis and found that, in addition to GART, two other purine pathway activities, phosphoribosylamine-glycine ligase (phosphoribosylglycinamide synthetase, GARS) and phosphoribosylformylglycinamidine cyclo-ligase (phosphoribosylaminoimidazole synthetase, AIRS), are similarly overproduced. All three activities are present together on the larger overproduced protein. A smaller protein appears to possess only GARS activity. Therefore, alternative mRNA processing can allow cells to produce enzyme activities in forms that are either linked or unlinked to other activities.

Gamma and gamma' chains of human fibrinogen are produced by alternative mRNA processing.

cDNAs and the genomic DNA coding for the gamma and gamma' chains of human fibrinogen have been isolated and characterized by sequence analysis. The cDNAs coding for the gamma and gamma' chains share a common nucleotide sequence coding for the first 407 amino acid residues in each polypeptide chain. The predominant gamma chain contains an additional four amino acids on its carboxyl-terminal end (residues 408-411). These four amino acids, together with the 3' noncoding sequences, are encoded by the tenth exon. Removal of the ninth intervening sequence following the processing and polyadenylation reactions yields a mature mRNA coding for the predominant gamma chain. The less prevalent gamma' chain contains 20 amino acids at its carboxyl-terminal end (residues 408-417). These 20 amino acids are encoded by the immediate 5' end of the ninth intervening sequence. This results from an occasional processing and polyadenylation reaction that occurs within the region normally constituting the ninth intervening sequence. Accordingly, the gene for the gamma chain of human fibrinogen gives rise to two mRNAs that differ in sequence on their 3' ends. These mRNAs code for polypeptide chains with different carboxyl-terminal sequences. Both of these polypeptides are incorporated into the fibrinogen molecule present in plasma.